CN101616685B - Unstructured recombinant polymers and uses thereof - Google Patents

Abstract

The present invention provides unstructured recombinant polymers (URPs) andproteins containing one or more of the URPs. The present invention also provides microproteins, toxins and other related proteinaceous entities, as well as genetic packages displaying these entities. The present invention also provides recombinant polypeptides including vectors encoding the subject proteinaceous entities, as well as host cells comprising the vectors. The subject compositions have a variety of utilities including a range of pharmaceutical applications.

Description

Unstructured recombinant polymers and its application

Cross reference

The application requires to submit on March 6th, 2006 priority of U.S. Provisional Application number 60/743,410, includes this application in this paper as a reference.The application is 11/528 of JIUYUE in 2006 submission on the 27th, 927 and 11/528,950 part continuation application, they require the 9/27/2005 provisional application serial number of submitting to 60/721,270,60/721,188 and 03,/21,/06 60/743,622 the priority of submitting to is included their full text in this paper as a reference.

Background of invention

Existing bibliographical information can improve some character of albumen by hydrophilic polymer is connected with albumen, particularly plasma clearance and immunogenicity (Kochendoerfer, G. (2003) Expert Opin Biol Ther, 3:1253-61), (Greenwald, R.B. etc. (2003) Adv Drug Deliv Rev, 55:217-50), (Harris, J.M. etc. (2003) Nat Rev Drug Discov, 2:214-21).The polymer-modified proteic example that is used for the treatment of patient by the FDA approval is A Dajin (Adagen), high Caspar (Oncaspar), PEG-intron, Pai Luoxin (Pegasys), Suo Mawo (Somavert) and Niu Lasita (Neulasta).More polymer-modified albumen is in the clinical trial.These polymer reduce kidney with respect to the hydrodynamic radius (being also referred to as stokes radius) of unmodified protein and filter clearance rate by increasing modified protein, thus bring into play usefulness (Yang, K. etc. (2003) Protein Eng, 16:761-70).In addition, polymer connects the interaction that can reduce modified protein and other albumen, cell or surface.Specifically, thus polymer connects and can reduce modified protein and immune antibody and other component interaction reduction host immunne response to modified protein.Outstanding interested is by PEGization, promptly connect linearity or the branch polyethylene glycol polymer comes modified protein.Immunogenic example of PEGization reduction such as PAL (Gamez, (2005) Mol Ther such as A., 11:986-9), antibody (Deckert, (2000) Int J Cancer such as P.M., 87:382-90.), Sbphylokinase (Collen, D. etc. (2000) Circulation, 102:1766-72) and hemoglobin (Jin, (2004) ProteinPept Lett such as C., 11:353-60).Usually, behind the purification unmodified protein, this base polymer is connected with proteins of interest by the chemical modification step.

Multiple polymers can be connected with albumen.Outstanding interested is the hydrophilic polymer with variable conformation and fine hydration in aqueous solution.Polymer commonly used is Polyethylene Glycol (PEG).Relative its molecular weight, these polymer be tending towards having bigger hydrodynamic radius (Kubetzko, S. etc. (2005) Mol Pharmacol, 68:1439-54).The connected albumen of the polymer that is connected is tending towards having limited interaction, and therefore polymer-modified albumen can keep its correlation function.

Polymer and proteinic chemical coupling need complicated multistep process.Typically, protein component must produce before the chemical coupling step and purification.Coupling step can cause producing the isolating product mixtures of needs, can cause that product significantly loses when separating.Perhaps, this type of mixture can be used as final drug products.Some examples of listing comprise PEGization interferon-' alpha ' product (Wang, B.L. etc. (1998) J Submicrosc Cytol Pathol, the 30:503-9 that uses with form of mixtures at present; Dhalluin, C. etc. (2005) BioconjugChem, 16:504-17).This type of mixture is difficult to produce to be identified, and comprises the isomer that reduces or do not have therapeutic activity.

The method that allows locus specificity to increase polymer such as PEG had been described.The selectivity PEGization of the unique glycosylation site of example such as target egg or engineering is built into the selectivity PEGization of the alpha-non-natural amino acid of target protein.In some cases can be by the terminal PEGization of avoiding the target protein lysine side-chain of careful control reaction condition selectivity PEGization protein N.And the method for another target protein locus specificity PEGization is to introduce alternative link coupled cysteine residues.All these methods all have tangible limitation.The selectivity PEGization of N-terminal needs careful process control and is difficult to eliminate side reaction.But introducing is used for the production of cysteine interferencing protein and/or the purification of PEGization.Specificity is introduced alpha-non-natural amino acid needs the specific host organism to be used for protein production.Another limitation of PEGization is that common PEG produces with the polymeric blends form, and these mixture length are similar but inconsistent.Other chemical polymerization thing also has same limitation.

Utilize the chemical coupling of multifunctional polymer to allow synthetic product with a plurality of albumen modules, this coupling ratio polymer and single protein structure domain coupling are more complicated.

Recently some protein of observing Pathogenic organisms contain the repetition peptide sequence, these sequences as if cause comprising the protein serum half-life of these sequences relatively long (Alvarez, P. etc. (2004) JBiol Chem, 279:3375-81).Show that also oligomerization sequence and other albumen fusion can causing serum half-life with the repetitive sequence of deriving based on this type of pathogen increase.Yet the oligomer in these pathogen sources has some defectives.The sequence in pathogen source is tending towards that immunogenicity is arranged.Also having to describe claims these sequences of modification can reduce its immunogenicity.Yet the Shang Weiyou report is attempted removing t cell epitope from the sequence that participates in immunoreation formation.And, require sequence to have good solubility in the pharmaceutical applications and to the low-down affinity of other target protein, and the sequence of not resisting former source according to this kind pharmaceutical applications as yet is optimized.

Therefore, need the compositions and the method that can allow a plurality of polymer modules are become with a plurality of protein module combinations defined Multidomain product badly.

Summary of the invention

The invention provides the unstructured recombinant polymers (URP) that contains at least 40 contiguous amino acids, wherein said URP substantially can not with the serum albumin non-specific binding, and wherein the glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) the residue total amount that comprise of (a) URP surpasses about 80% of URP total amino acid content; And/or (b) the Chou-Fasman algorithm is measured at least 50% aminoacid and is lacked secondary structure.In related embodiment, the invention provides a kind of unstructured recombinant polymers (URP) that comprises at least 40 contiguous amino acids, the external serum degradation half life of wherein said URP is longer than about 24 hours, and the total amount of the glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residue that are wherein comprised among (a) URP surpasses about 80% of URP total amino acid content; And/or (b) the Chou-Fasman algorithm is measured at least 50% aminoacid and is lacked secondary structure.Theme URP can comprise the alpha-non-natural amino acid sequence.Desired is, selects URP to be used to mix heterologous protein, in case after URP mixes heterologous protein, compare with the corresponding protein that does not mix described URP, described heterologous protein shows long serum secretion half-life and/or higher dissolubility.Half-life can prolong 2 times, 3 times, 5 times, 10 times or longer.In some instances, URP mixes heterologous protein and causes that the apparent molecular weight through size exclusion chromatograph estimation improves at least 2 times, 3 times, 4 times, 5 times or more.In some instances, the T epi-position of URP scoring less than-3.5 (as ,-4 or lower ,-5 or lower).In some instances, URP can mainly comprise hydrophilic residue.Desired is that the Chou-Fasman algorithm is measured at least 50% aminoacid and lacked secondary structure.The glycine residue that comprises among the URP accounts at least 50% of total amino acids that URP comprises.In some instances, be contained in the arbitrary type aminoacid that is selected from glycine (G), agedoite (D), alanine (A), serine (S), threonine (T), glutamic acid (E) or proline (P) among the URP and account for the content of URP total amino acids alone greater than about 20%, 30%, 40%, 50%, 60% or higher.In some instances, URP comprises greater than about 100,150,200 or more a plurality of contiguous amino acid.

The present invention also provides the protein that contains one or more theme URP, and wherein theme URP and protein are allos.Assemble the URP total length and can surpass about 40,50,60,100,150,200 or more a plurality of aminoacid.This albumen can comprise one or more functional modules, and functional module can be selected from effect module, binding modules, N-terminal module, C-terminal module or its combination in any.Desired is, theme albumen contains a plurality of binding modules, and wherein single binding modules has binding specificity for same or different target spots.Binding modules can comprise the support that contains disulphide that is formed by cysteine pairing in the support.Binding modules can combine with the target molecule that is selected from cell surface protein, secretory protein, plasmosin or nucleoprotein.Target spot can be ion channel and/or GPCR.Yes desired, the effect module can be toxin.The proteic half-life of containing theme URP usually grows 2,3,4,5,10 or more many times than the corresponding albumen that does not contain described URP.

In embodiment independently, the invention provides the protein of the non-natural generation that comprises at least 3 aminoacid sequence recurring units, each recurring unit comprises at least 6 aminoacid, wherein has a plurality of sections of about 6-15 the contiguous amino acid that comprises at least three recurring units in one or more natural human albumen.On the one hand, exist a plurality of sections or each to include the section of 9-15 the contiguous amino acid of having an appointment in recurring unit in one or more natural human albumen.This section can comprise about 9-15 aminoacid.Three recurring units can have remarkable sequence homology, as working as comparison time series homogeneity greater than about 50%, 60%, 70%, 80%, 90% or 100%.This non-natural albumen also can comprise one or more modules that is selected from binding modules, effect module, multimerization module, C-terminal module or N-terminal module.Desired is that non-natural protein can comprise the single recurring unit with theme unstructured recombinant polymers (URP).

The present invention also provides the recombination of polynucleotide that comprises coding theme URP, the protein that contains URP, microprotein and toxin.The present invention also provides the carrier that contains the theme polynucleotide, the host cell that carries this carrier, shows heredity bag, the protein that contains URP, toxin or other arbitrary protein entity disclosed herein of theme URP.This paper also provides the selectivity library of expression vector of the present invention.

The present invention also provides the method that comprises unstructured recombinant polymers (URP) that produces.Described method comprises that (i) provides the host cell of the recombination of polynucleotide that contains encoding said proteins, described albumen contains one or more URP, described URP contains at least 40 contiguous amino acids, wherein said URP substantially can not with the serum albumin non-specific binding, and wherein among (a) URP the total amount of contained glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residue surpass about 80% of URP total amino acids amount; And/or (b) the Chou-Fasman algorithm is measured at least 50% aminoacid and is lacked secondary structure; (ii) under appropraite condition, cultivate described host cell in the suitable culture medium, so that express described albumen by described polynucleotide.The suitable host cell is eucaryon (as Chinese hamster ovary celI) and prokaryotic cell.

The present invention also provides and prolongs the protein serum method of secretion half-life, comprise: described protein and one or more unstructured recombinant polymers (URP) are merged, wherein, URP comprises at least 40 contiguous amino acids, and wherein the total amount of contained glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residue surpasses about 80% of URP total amino acids amount among (a) URP; And/or (b) the Chou-Fasman algorithm is measured at least 50% aminoacid and is lacked secondary structure; Described URP substantially can not with the serum albumin non-specific binding.

The present invention also provides between detection heredity extrinsic protein that bag is showed and the target spot whether have the synergistic method of specificity, wherein said albumen comprises one or more unstructured recombinant polymers (URP), and described method comprises: (a) provide and show the proteic heredity bag that comprises one or more unstructured recombinant polymers (URP); (b) the heredity bag is contacted with target spot; (c) detect the formation that stabilize proteins-target spot complex is wrapped in heredity, thus the synergistic existence of detection specificity.Described method can comprise that also acquisition is from the proteic nucleotide sequence of the encoding exogenous of heredity bag.In some instances, at URP with comprise and exist between the target spot of serum albumin or do not exist specificity to interact.In some instances, at URP with comprise and exist between the target spot of serum albumin enzyme or do not exist specificity to interact.

The present invention also comprises the heredity bag of showing microprotein, and wherein said microprotein keeps the binding ability of target spot natural with it.In some respects, microprotein is showed the binding ability with at least one ion channel family, and described ion channel family is selected from sodium, potassium, acetylcholine or chloride channel.Desired is, described microprotein be ion channel in conjunction with microprotein, and modified the microprotein ratio with corresponding unmodified with (a), can be in conjunction with different passage family; (b) with the microprotein ratio of corresponding unmodified, the different subfamily combinations of described microprotein and same passage family; (c) with the microprotein ratio of corresponding unmodified, described microprotein combines with the variety classes of same passage subfamily; (d) with the microprotein ratio of corresponding unmodified, described microprotein combines with the different loci of same passage; And/or (e) with the microprotein ratio of corresponding unmodified, described microprotein combines with the same site of same passage, but produces different biological effects.In some aspects, described microprotein is a toxin.The present invention also provides the heredity bag library of showing theme microprotein and/or toxin.Desired is that described heredity bag is showed the proteotoxin of retaining part or whole toxicity spectrums.This toxin can derive from single toxin protein, or derived from toxin family.The present invention also provides a kind of heredity bag library, and toxin family is showed in wherein said library, wherein said family retaining part or whole native toxicity spectrums.

The present invention also provides and has comprised the albumen of a plurality of ion channels in conjunction with the territory, wherein the single structure territory is the microprotein domain, this microprotein domain is modified the microprotein domain ratio with corresponding unmodified with (a), can be in conjunction with different passage family; (b) with the microprotein domain ratio of corresponding unmodified, the different subfamily combinations of described microprotein domain and same passage family; (c) with the microprotein domain ratio of corresponding unmodified, described microprotein domain combines with the variety classes of same passage subfamily; (d) with the microprotein domain ratio of corresponding unmodified, described microprotein domain combines with the different loci of same passage; (e) with the microprotein domain ratio of corresponding unmodified, described microprotein domain combines with the same site of same passage, but produces different biological effects; And/or (f) with the microprotein domain ratio of corresponding unmodified, the same site of described microprotein domain and same passage in conjunction with and produce identical biological effect.

The present invention also relates to obtain the method for the microprotein with required character, described method comprises that (a) provides the theme library; (b) screening selectivity library is to obtain the phage that at least a displaying has the microprotein of required character.The polynucleotide, carrier, heredity bag and the host cell that use in arbitrary disclosed method also are provided.

It is for referencial use to include this paper in

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Brief Description Of Drawings

New feature of the present invention is specifically listed by appended claims.The illustrated embodiment of the use principle of the invention that following detailed description is listed and accompanying drawing will make the reader that characteristics of the present invention and advantage are had better understanding, and accompanying drawing is as follows:

Fig. 1 shows the modular assembly of MURP.Binding modules, effect module and multimerization module are represented with circle.URP module, N-terminal and C-terminal module are represented with rectangle.

Fig. 2 shows MURP modular structure example.Binding modules (BM) among MURP can have identical or different target spot specificity.

Fig. 3 shows that the repetitive proteins based on the human sequence can comprise the amino acid sequence, and this sequence can comprise t cell epitope.These new sequences form in the junction of adjacent repetitive.

Fig. 4 shows the URP sequential design based on the repetitive proteins of three people's donor sequences D1, D2 and D3.Select the repetitive of this URP, even (even) 9-aggressiveness sequence of crossing over the adjacent cells connection can be found at least a people's donor sequences.

Fig. 5 is the example of conduct based on the URP sequence of the repetitive proteins of three people's protein sequences.The figure below shows that all 9-aggressiveness subsequences appear in a kind of people's donor albumen at least among the URP.

Fig. 6 is based on the example of the URP sequence of people POU domain residue 146-182.

Fig. 7 shows by insert the advantage that the URP module is separated the module that contains rich information sequence between sequence.Left figure demonstration directly obtains new sequence with modules A and B fusion at join domain.These catenation sequences can be epi-position.Right figure is presented between modules A and B and inserts the URP module and can prevent to form this type of catenation sequence that comprises from the partial sequence of modules A and B.The substitute is, the end of modules A and B produces the catenation sequence that comprises the URP sequence, therefore predicts that it has low immunogenicity.

Fig. 8 demonstration is sent construction based on the medicine of URP.Drug molecule that hexagon is represented and MURP chemical coupling.

Fig. 9 shows the MURP that comprises the responsive site of protease.Design URP module makes its blocking effect functions of modules.The protease cutting removes the part of URP module and causes the active raising of effector function.

Figure 10 show the URP module how at binding modules and effect intermodule as joint.Binding modules can cause that the local concentration of effect module raises with targeted integration and near target spot.

Figure 11 shows from the process of the gene of short URP module library construction coding URP sequence.URP module library can be inserted in the filling carrier that contains green fluorescent protein (GFP), green fluorescent protein (GFP) helps the identification of high expressed URP sequence as reporter.Can pass through repeatedly the encoding gene that dimerization makes up long URP sequence as shown in the figure.

Figure 12 shows the MURP that contains a plurality of death receptor binding modules.Trimerizing triggers death receptor, and therefore, at least three MURP in conjunction with original paper that contain a death receptor can especially effective inducing cell death.The below of figure shows can improve the specificity of MURP for illing tissue by adding one or more tumor tissues specificity binding modules.

Figure 13 shows the MURP that contains four specific for tumour antigen binding modules (rectangle) and effect module such as interleukin-22.

Figure 14 shows the flow chart that makes up the URP module that contains 288 residues.Make up the fusion rotein of URP module and GFP.At first structure contains 36 amino acid whose URP module libraries, then passes through repeatedly dimerization and produces 288 amino acid whose URP modules (rPEG_H288 and rPEG_J288).

The aminoacid and the nucleotide sequence of 88 amino acid whose URP modules of Figure 152 (rPEG_J288).

The aminoacid and the nucleotide sequence of 88 amino acid whose URP modules of Figure 162 (rPEG_H288).

The aminoacid sequence of the rich serine sequence area of Figure 17 people's DSPP.

Figure 18 shows MURP derivant bank.Protein comprises two cysteine residues that can form weak SS bridge.Process this albumen, keep the SS bridge complete.Formulated is also injected to patient to go back ortho states.It can oxidation also form the very limited heavy polymer of diffusion the injection back near injection site.By conditional proteolysis or the reduction of conditional SS cross-bond, active MURP is slowly leached from injection site.

Figure 19 shows the depot forms of MURP.Very limited in injection site MURP diffusion, by conditional proteolysis it is discharged from injection site.

Figure 20 shows the depot forms of the MURP that contains rich histidine sequence.With MURP with contain the insoluble pearl injection formulated together of solidifying nickel.Combine and slowly discharge into circulation with nickel bead at injection site MURP.

Figure 21 shows the MURP that comprises the multimerization module.Last figure shows the MURP that comprises a dimerization sequence.The result is that it forms the effectively dimer of its molecular weight of multiplication.Middle figure shows the design of three MURP that comprise two multimerization sequences.This MURP can form the polymer with very high effective molecular weight.Figure below shows the MURP comprise a plurality of RGD sequences, and known RGD sequence can be in conjunction with cell surface receptor, thus prolong half-life.

Figure 22 shows the multiple MURP that is designed for blocking-up or regulates ion channel function.Circle is represented the specific binding modules of ion channel.These binding modules originate from or are identical with natural toxin, have the ion channel receptor affinity.Can add other in conjunction with the territory at ion channel specificity binding modules either side as shown in the figure, thereby improve effect or the specificity of MURP a certain cell type.

Figure 23 shows several MURP designs of prolong half-life.Can be by increasing chain length (A), chemical multimerization (B), in molecule, adding the binding modules (C) of the separated multicopy in non-binding site, (D E), comprises that multimerization sequence (F) increases effective molecular weight to make up the chemical multimer of similar C.

Figure 24 shows can be by the MURP that binding modules and the chemical coupling of reorganization URP sequence are formed.Design this URP sequence, to comprise a plurality of lysine residues (K) as the coupling site.

Figure 25 shows the design in 2SS binding modules library.Comprise constant 1SS sequence in the middle of this sequence, 1SS sequence and the random sequence side joint that contains cysteine apart from 1SS core different distance.

Figure 26 shows the design in 2SS binding modules library.Comprise constant 1SS sequence in the middle of this sequence, 1SS sequence and the random sequence side joint that contains cysteine apart from 1SS core different distance.

Figure 27 shows the design in 1SS binding modules dimer library.At first, react the set of amplification ISS binding modules by twice PCR.Merge the PCR product that obtains and in follow-up PCR step, produce dimer.

Figure 28 is presented at hatched in 50% mice serum at the most after three days, and the Western that contains the fusion rotein of 288 aminoacid URP sequence rPEG_J288 analyzes.

Figure 29 shows that the combination of the preexisting antibody that resists 288 amino acid whose URP sequences detects result of the test.

Figure 30 shows that the MURP that comprises (monomer), two (dimer), four (tetramer) or do not have (rPEG36) binding modules is combined by the specificity of the VEGF on microtitration plate with bag.

Figure 31 shows the aminoacid sequence that EpCAM is had specific MURP.This sequence comprises four binding modules (underscore) that EpCAM had affinity.This sequence comprises the N-terminal Flag sequence of only two lysines in the full sequence.

Figure 32 shows the design in the additional library of ISS.1SS module at random can be added the N or the C-terminal of preliminary election binding modules or adds two ends simultaneously.

Figure 33 shows the comparison of three finger-like toxin correlated serieses.This figure also shows the 3D structure that NMR resolves.

Figure 34 shows the library design based on three finger-like toxin.X is residue at random.Indicate the codon selection of each random site.

Figure 35 shows the comparison of plexin correlated series.

Figure 36 shows the library design based on plexin.X is residue at random.Indicate the codon selection of each random site.

Figure 37 shows the sequence that DR4, ErbB2 and HGFR is had the relevant binding modules of specific pexin.

Figure 38 shows has the specific combination test in conjunction with the territory based on microprotein to VEGF.

Figure 39 shows has specific separation from the 2SS and the 3SS binding modules sequence that make up (buildup) library to VEGF.Last figure shows the proteinic PAGE glue analysis of pyrolysis purification.

Figure 40 shows the clone's step that makes up URP sequence rPEG_J72.

Figure 41 shows to make up to have 36 amino acid whose URP module libraries that are called rPEG_J36.With the shorter section of three coding rPEG_J12 and stop module and assemble and become the rPEG_J36 coding region.

Figure 42 shows the nucleotide sequence and the translation of filling carrier pCW0051.The fill area side joint is in BsaI and BbsI site, and comprises a plurality of termination codoies.

Figure 43 shows the PAGE glue of the purification of the URP rPEG_J288 that merges with GFP.Swimming lane 2 is a cell lysate; Swimming lane 3:IMAC purified product; Swimming lane 4: anti--the Flag purified product.

The aminoacid sequence of Figure 44 rPEG_J288 and people's effector domain interferon-ALPHA, G-CSF and human growth hormone's fusion rotein.

Figure 45 shows that the Western of the expressing fusion protein of rPEG_J288 and human growth hormone's (swimming lane 1 and 2), interferon-ALPHA (swimming lane 3 and 4) and GFP (swimming lane 5 and 6) analyzes.Analyze each proteic solubility and insoluble material.

Figure 46 shows the design based on the MURP of toxin OSK1.Can add URP sequence and/or binding modules at the OSK1 either side as shown in the figure.

Figure 47 has described the exemplary products form that contains theme URP.

Detailed Description Of The Invention

Although this paper has described preferred implementation of the present invention, those skilled in the art should be understood that this type of embodiment only provides explanation as an example.Those skilled in the art can make different variations, change and substitute not deviating under the prerequisite of the present invention.Should be understood that the alternative arrangement that in the middle of enforcement the present invention, can use multiple embodiment described herein.This paper is intended to utilize appended claims to define scope of the present invention, the method and structure in these claim scopes with and the equivalent form of value therefore also be capped.

General technology:

Unless indicate in addition, the invention process adopts routine immunization, biochemistry, chemistry, molecular biology, microbiology, cytobiology, genomics and recombinant DNA technology, and these are all within those skilled in the art's skill.Referring to Sambrook, Fritsch and Maniatis, MOLECULARCLONING:A LABORATORY MANUAL (molecular cloning: laboratory manual), second edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (newly organized molecular biology method) (volume such as F.M.Ausubel, (1987)); METHODS IN ENZYMOLOGY (Enzymology method book series) (academic press (Academic Press)): PCR2:A PRACTICAL APPROACH (PCR2: (M.J.MacPherson hands-on approach), B.D.Hames and G.R.Taylor compile (1995)), Harlow and Lane, compile (1988) ANTIBODY, A LABORATORY MANUAL (antibody, laboratory manual) and ANIMAL CELL CULTURE (animal cell culture) (R.I.Freshney, compile (1987)).

Definition:

The singulative that uses in description and claims " one ", " a kind of " and " one " comprise its plural form, unless context has indication in addition.For example, term " cell " comprises the plural form of cell, and composition thereof.

Term " polypeptide ", " peptide ", " aminoacid sequence " and " protein " are used interchangeably herein, refer to the random length polymer of amino acid.This polymer can be linearity or branch, can comprise modified amino acid, also can be interrupted by non-aminoacid.The amino acid polymer of modification also contained in this term, for example, forms disulfide bond, glycosylation, fatization, acetylation, phosphorylation or other any operation, as with the link coupled polymer of marker components.Term used herein " aminoacid " refers to natural and/or non-natural or synthetic aminoacid, includes but not limited to glycine and D or L optical isomer, amino acid analogue and peptide mimics.Use standard list or trigram coded representation aminoacid.

" repetitive sequence " is to be described to repetition peptide sequence, formation directly repetition or oppositely repetition, or the alternately repeated oligomer of multiple sequence motifs.Mutually the same or the homology of these multiple oligomerization sequences, but multiple repetition motif also can be arranged.The feature of repetitive sequence is that information content is extremely low.Repetitive sequence is not the required characteristics of URP, preferred in some cases non repetitive sequence.

Can characterize aminoacid according to amino acid whose hydrophobicity.Several range scales have been developed.The example of a range scale is by Levitt, exploitation such as M (referring to Levitt, M (1976) J Mol Biol 104,59, #3233 is listed in Hopp, TP etc. (1981) Proc Natl Acad Sci USA 78,3824, #3232).The example of " hydrophilic amino acid " is arginine, lysine, threonine, alanine, agedoite and glutamine.Outstanding interested hydrophilic amino acid is aspartic acid, glutamic acid, serine and glycine.The example of " hydrophobic amino acid " is tryptophan, tyrosine, phenylalanine, methionine, leucine, isoleucine and valine.

A kind of state of peptide in solution described in term " degeneration conformation ", it is characterized in that the conformational freedom of peptide main chain is very big.Most of peptides and protein change the degeneration conformation into when high concentration denaturant or elevated temperature.The peptide that is in the degeneration conformation has feature CD spectrum, is characterised in that the interaction that lacks long scope when detecting as NMR usually.The degeneration conformation and not folded conformation be synonym.

Term " destructuring protein (UNP) sequence " and " unstructured recombinant polymers " (URP) are used interchangeably herein.These two terms refer to total denatured polypeptide sequence, as having the aminoacid sequence of the typical behavior of similar denatured polypeptide sequence under the physiological condition that describes in detail at this paper.The URP sequence does not have definite tertiary structure, and has limited secondary structure or do not have secondary structure through the detection of Chou-Fasman algorithm.

The plasma membrane component of term used herein " cell surface protein " phalangeal cell contains integration and the memebrane protein periphery, glycoprotein, polysaccharide and the lipid of forming plasma membrane.Conformity membrane albumen is for crossing over the transmembrane protein that the cytoplasma membrane double-layer of lipoid extends.A typical conformity membrane albumen comprises the transmembrane segment that at least one contains hydrophobic amino acid residue.Peripheral memebrane protein does not extend into the double-layer of lipoid hydrophobic interior, directly or indirectly is incorporated into the film surface by the covalently or non-covalently interaction with other membrane component.

The term " film ", " Cytoplasm ", " nucleus " and " secretion " that are applied to cell protein refer in particular to the outer and/or Subcellular Localization of maximum, the main or preferred born of the same parents of this cell protein.

" cell surface receptor " representative can with its subgroup of the bonded memebrane protein of part separately.Cell surface receptor is the molecule that is anchored on the cytoplasma membrane or inserts in the cytoplasma membrane.They have formed a big albumen, glycoprotein, polysaccharide and lipid family, and not only as the structural constituent of plasma membrane, also the controlling element of various biological function is managed in conduct for they.

A part of term " module " finger protein, this part are physically or be different from the other parts of this albumen or peptide on the function.A module can comprise one or more domains.Usually, module or domain can be the single stable three dimensional structure of irrelevant size.The tertiary structure in typical structure territory is keeping stable and is remaining unchanged when separating or merging with other domain covalency in the solution.Usually, domain has the specific tertiary structure that is formed by secondary structure spatial relationship (as β-lamella, alpha-helix and destructuring ring).In microprotein family structure territory, disulphide bridges normally determines the main element of tertiary structure.In some instances, domain is to produce some specific function activity, as affinity (to a plurality of binding sites of same target spot), polyspecific (to the binding site of different target spots), half-life (using domain, cyclic peptide or a linear peptides), with serum albumin such as human serum albumin (HSA) or IgG (hIgG1,2,3 or 4) or the bonded module of erythrocyte.Function determines that domain has the unique biological function.For example, the ligand binding domain of receptor is the domain of binding partner.Antigen binding domain refers to the part of antigen combining unit or the antibody of conjugated antigen.Function determines that domain need not to be encoded by contiguous amino acid sequence.Function determines that domain can comprise the domain that one or more physics are determined.For example, receptor is divided into the outer ligand binding domain of born of the same parents, membrane spaning domain and born of the same parents' internal effect domain usually." film anchoring structure territory " refers to mediate membrane-bound protein part.Usually, film anchoring structure territory is made up of hydrophobic amino acid residue.Perhaps, film anchoring structure territory can comprise modified amino acid, as the aminoacid that is connected with fatty acid chain, and then this albumen is anchored on the film.

Be used for proteic " non-natural produces " and refer to comprise the amino acid whose albumen that at least one is different from corresponding wild type or native protein.Carry out blast search and detect the non-natural sequence, for example be the length of sequence interested (search sequence) and use BLAST2.0 to use minimum minimum probability and execution blast search relatively the time with Genbank nonredundancy (" nr ") data base when comparison window.Altschul etc. have described BLAST 2.0 algorithms respectively in (1990) J.Mol.Biol.215:403-410.NCBI (National Center for Biotechnology Information) provides to the public and carries out the software that BLAST analyzes.

" host cell " comprises the cell individual or the cell culture that can be or become to be the theme the carrier acceptor.Host cell comprises the offspring of single host cell.Since natural, unexpected or deliberate sudden change, described offspring not necessarily identical (on the form or on total DNA genome) with the original parent cell.Host cell comprises and utilizes cells transfected in the carrier body of the present invention.

Term used herein " separation " refers to from cell or other component under the separating natural state polynucleotide, peptide, polypeptide, protein, antibody or its fragment to association.Those skilled in the art understand that polynucleotide, peptide, polypeptide, protein, antibody and fragment thereof that non-natural produces need not to come the homologue of generation natural with it to be distinguished by " separation ".In addition, " spissated ", " isolating " or " dilution " polynucleotide, peptide, polypeptide, protein, antibody or its fragment can generation natural with it homologue distinguished because the concentration of its every volume or molecular number are greater than " spissated " or less than the homologue of " isolating " its natural generation.

" connection " and " fusion " or " fusion " herein is used interchangeably.These terms refer to comprise that by any-mode chemical coupling or recombination form link together plural chemical component or component." frame endomixis " refers to that two or more open reading frame (OFR) are connected to form long continuously OFR in the mode that keeps original OFR proper reading frame.Therefore, resulting recombination fusion protein is an albumen that comprises two or more sections (these sections so do not connect under natural situation usually) of corresponding original OFR coded polypeptide.

When context was polypeptide, " linear order " or " sequence " referred to the amino amino acid sequence that arrives the direction of carboxyl terminal in polypeptide, and residue adjacent one another are is the contiguous nucleotide sequence of this polypeptide primary structure in the sequence." partial sequence " refers to the known linear order that contains the polypeptide portion of extra residue in one or two direction.

" allos " refers to compare with this entity remainder, derived from the entity of different genotype.For example, one section rich glycine sequence is removed from its natural coded sequence and it is connected with coded sequence operability except that this natural coded sequence then obtains the rich glycine sequence of allos.The term " allos " that is applied to polynucleotide, polypeptide refers to compare with this entity remainder, and these polynucleotide or polypeptide are derived from the entity of different genotype.

Term " polynucleotide ", " nucleic acid ", " nucleotide " and " oligonucleotide " are used interchangeably.They refer to the nucleotide of random length, or the polymerized form of deoxyribonucleotide, ribonucleic acid or its analog.Polynucleotide can have any three dimensional structure, and can carry out known or unknown any function.Classify the non-limitative example of polynucleotide down as: RNA, nucleic probe and the primer of the locus of the coding of gene or genetic fragment or noncoding region, linkage analysis definition, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombination of polynucleotide, branch's polynucleotide, plasmid, carrier, the DNA of isolating arbitrary sequence, isolating arbitrary sequence.Polynucleotide can comprise the nucleotide of modification, as methyl nucleotide and nucleotide analog.If exist, can modified nucleotide before the polymer assembling or after the assembling.The non-nucleotide component can interrupt nucleotide sequence.Can after polymerization, further modify polynucleotide, as with the marker components coupling.

" reorganization " that is applied to polynucleotide refers to that polynucleotide are combination product of various clones, restriction enzyme digestion and/or Connection Step and other step, and these steps produce the construction that is different from the polynucleotide that occurring in nature finds.

Term " gene " or " genetic fragment " are used interchangeably herein.They refer to contain at least one open reading frame at the polynucleotide of transcribing and translating back codified specific protein.As long as polynucleotide comprise at least one and cover whole coding region or its segmental open reading frame, then this gene or genetic fragment can be genomic DNA or cDNA." fusion gene " is a gene that comprises at least two heterologous polynucleotide that link together.

" carrier " is to insert that nucleic acid molecules is transported in the host cell or to the nucleic acid molecules of the preferred self replication between the host cell.This term comprise major function be with DNA or RNA insert the carrier of cell, replicating vector and the major function that major function is DNA or rna replicon is the expression vector of DNA or rna transcription and/or translation.Also comprise the carrier that more than one above-mentioned functions are provided." expression vector " is the polynucleotide that can be transcribed and translate into polypeptide when introducing the suitable host cell." expression system " is often referred to the suitable host cell that comprises expression vector that can be used for producing required expression product.

When context is MURP, " target spot " be binding modules or URP johning knot compound module can in conjunction with, and binding events causes the chemical molecular or the structure of required biologic activity.Target spot can be by this albumen inhibition, activation or the protein ligands or the receptor that start.The example of target spot is that hormone, cytokine, antibody or antibody fragment, cell surface receptor, kinases, somatomedin and other have the chemical constitution of biologic activity.

" functional module " can be any non-URP in the protein product.Therefore functional module can be binding modules (BM), effect module (EM), multimerization module (MM), C-terminal module (CM) or N-terminal module (NM).Usually, the feature of functional module is the high information content of its aminoacid sequence, and promptly they comprise in many different aminoacid and these aminoacid a lot important for the function of functional module.Functional module has secondary and tertiary structure usually, can be a folded protein domain and can comprise 1,2,3,4,5 or more a plurality of disulfide bond.

Term " microprotein " refers to the classification among the SCOP data base.Microprotein normally has the minimum albumen of fixed structure, usually but definitely do not contain few to 15 aminoacid and two disulfide bond, or 200 aminoacid of as many as and more than 10 disulfide bond.Microprotein can contain one or more microprotein domains.Different disulfide bond types causes some microprotein domains or domain family can have a plurality of stable differences structure different with a plurality of similaritys, therefore uses term to stablize with relative mode and distinguishes microprotein and polypeptide and non-microorganism protein structure domain.The most of microbe proteotoxin is made of the single structure territory, but the cell surface receptor microprotein usually has a plurality of domains.Microprotein can be so little, because by disulfide bond and/or ion such as calcium, magnesium, manganese, copper, zinc, ferrum or multiple other multivalent ion, but not common hydrophobic core makes it folding stable.

Term " support " refers to when making up the albumen library as minimum polypeptide " framework " or " sequence motifs " of guarding consensus sequence.Variable and hypermutation position is between the fixing or conserved residues/position of support.Variable region between fixed support provides a large amount of different aminoacid to combine in order to provide with the specificity of target molecule.Support usually when comparing in sequence associated protein family observed conserved residues limit.Perhaps, folding or structure needs fixedly residue, particularly when comparing proteic function not simultaneously.Description for a microprotein support can comprise quantity, position or spacing, and the binding pattern of cysteine, and the position and the kind of arbitrary fixedly residue in the ring comprise the binding site of ion such as calcium.

" folding " of microprotein defined by disulfide bond link mode (being 1-4,2-6 and 3-5) to a great extent.This pattern is that topology is constant, and is difficult for being converted into other pattern usually when (as by reduction and oxidation (reductant-oxidant)) not disconnecting and reconnect disulfide bond.Usually, the native protein with correlated series adopts two identical sulfur to become the key pattern.Major decision bunch is cysteine distance mode (CDP) and some fixed non-cys residues and melts combine site (if existence).In example seldom, protein folding also is subjected to the influence of sequence (being propetide) on every side, by residue chemical derivatization (be γ-carboxylated) albumen is combined with bivalent metal ion (being calcium) in some instances and helps it folding.Concerning most microproteins, this type of folding help is unnecessary.

Yet based on the length and composition different of ring, these albumen with identical one-tenth key pattern also can comprise a plurality of folding, and described ring is enough big, to give this albumen diverse structure.An example is conotoxin, ring toxin (cyclotoxin) and anato domain family, and they have identical DBP but CDP is obviously different, are considered to different folding.The determiner of protein folding is with respect to the folding structure that greatly changes of difference, as any attribute of the difference (particularly folding the required retainer ring residue or the position or the composition of calcium (or other metal or cofactor) binding site) of the sequence motifs that encircles between the quantity of cysteine and the spacing that becomes key pattern, cysteine, cysteine.

Term " disulfide bond becomes the key pattern " or " DBP " refer to the connection mode of cysteine, are numbered to C-terminal from proteinic N-terminal with 1-n.It is that topology is constant that disulfide bond becomes the key pattern, means to utilize to untie one or more disulfide bond as the oxidoreduction condition and just can change it.The 0048-0075 section is listed possible 2-, 3-and is become the key pattern with the 4-disulfide bond.

Term " cysteine distance mode " or " CDP " refer to separate the number of the non-cysteine of cysteine on linear protein chain.Use multiple symbol: C5C0C3C equals C5CC3C and equals CxxxxxCCxxxC.

Term " position n6 " or " n7=4 " refer to encircle between cysteine, and " n6 " refers to the ring between C6 and C7; " n7=4 " refers to that the ring length between C7 and C8 is 4 aminoacid (disregarding cysteine).

Serum degradation-resistant-can eliminate protein by degraded in blood, this degraded is usually directed to the protease in serum or the blood plasma.By with albumen and people's (or suitable mice, rat, monkey) serum or blood plasma 37 ℃ down mixed different number of days (promptly 0.25,0.5,1,2,4,8,16 day) detect the serum degradation-resistant.Then, test the sample of these time points of electrophoresis and utilize antibody detection protein with Western.Antibody can be the antibody of albumen label.If albumen shows on western and injects the identical single band of albumen size, then do not have degraded and take place.The time point that Western detects 50% protein degradation is this proteic serum degradation half life.

Though serum albumin combination-MURP contains several and cell surface target spot and/or the bonded module of serum albumin usually, we expect that URP does not have the activity of non-expectation substantially.Should design URP and minimize and avoid and serum albumin, comprise the interaction (combination) of antibody.Utilize combining of the different URP design of ELISA screening and serum albumin, the fix blood albumin adds URP then, hatches the amount of the bonded URP of washing back detection.A kind of method is to utilize identification to add the antibody test URP of the label of URP, a kind of diverse ways is fixing URP (as by merging with GFP), add human serum, hatch, utilize the anti-human IgG of second antibody such as goat to detect the people's antibody amount that still is incorporated into URP after the washing.Utilize we URP of design of these methods to demonstrate very low-level serum albumin combination.Yet, in some applications, need combine with serum albumin or serum contactin, for example, because can further prolong the secretion half-life.In this case, can use identical experiment to design and serum albumin or serum contactin such as HSA or the bonded URP of IgG.In other cases, the binding modules that comprises through design and serum albumin or serum contactin such as HSA or the bonded peptide of IgG can be inserted among the MURP.

Unstructured recombinant polymers (URP):

One aspect of the present invention is the design of destructuring recombinant polymers (URP).When generation had the recombiant protein of treatment and/or diagnostic value, theme URP was particularly useful.Theme URP shows following one or more characteristics.

Theme URP contains the aminoacid sequence that has general character with the denatured polypeptide sequence usually under physiological condition.The URP sequence under physiological condition usually and the behavior of denatured polypeptide sequence similar.Under physiological condition, the URP sequence lacks the secondary and the tertiary structure of good qualification.This area has been set up secondary and the tertiary structure that several different methods is determined given polypeptide.For example, utilize the CD spectrum in " UV far away " spectrum district (190-250nm) to detect the secondary structure of polypeptide.Alpha-helix, beta sheet and the characteristic peak and the width of each self-forming CD spectrum of coiled structure at random.Can pass through some computer program and algorithm equally, (Chou, P.Y. etc. (1974) Biochemistry 13:222-45) determines secondary structure as the Chou-Fasman algorithm.For a given URP sequence, this algorithm can predict whether there are some or do not have secondary structure.Usually, because the secondary of low degree and tertiary structure, the URP sequence has the spectrum of similar degeneration sequence usually.Desired is can design URP to make it have the conformation of obvious degeneration under physiological condition.The URP sequence has the very conformation elasticity of high level usually under physiological condition, compare with the globulin of close molecular weight, and they are tending towards forming big hydrodynamic radius (Stokes radius).Physiological condition used herein refers to the condition of a series of simulation live body situations, comprises temperature, salinity, pH.Set up the physiology correlated condition that many experiment in vitro use.Usually, physiological buffer comprises the salt of physiological concentration and is adjusted to the about 6.5-7.8 of neutral pH scope, preferably about 7.0-7.5.Sambrook etc. (1989) enumerated several physiological buffers before, did not repeat them here.Physiology associated temperature scope is from about 25 ℃-38 ℃, preferred about 30 ℃-37 ℃.

Theme URP can be the reduced immunogenicity sequence.Reduced immunogenicity is the elastic direct result of URP sequence conformation.So-called comformational epitope in many antibody recognition proteantigens.Comformational epitope is formed by the protein surface zone of a plurality of discontinuous aminoacid sequences that contain proteantigen.Proteic accurately folding these sequences are become can be by the clear and definite particular configuration of antibody recognition.Design preferred URP to avoid forming comformational epitope.What for example, cherish a special interest is the URP sequence that seldom is tending towards forming fine and close folded conformation in aqueous solution.Specifically, by being chosen in the sequence of opposing antigen processing in the antigen presenting cell, select not obtain reduced immunogenicity with the sequence of MHC good combination and/or the sequence of selection derived from human sequence.

Theme URP can be the sequence with height protease resistant.Protease resistant also can be the elastic result of URP sequence conformation.Can be by avoiding known protein enzyme recognition site design protease resistant.Perhaps, can utilize phage display or correlation technique from selecting the protease resistant sequence at random or the half random sequence library.Need be used for special applications, during as slow release from the protein bank, the serum albumin enzyme action can be cut the site and be building up among the URP.That cherish a special interest is the URP that stablizes (as long serum half-life, difficult by the cutting of the protease in the body fluid) at the blood camber.

The feature of theme URP also is, when mixing albumen, compares with the corresponding albumen of no URP, and this albumen shows long half-life and/or higher dissolubility.[method of determining serum half-life is known in the art (referring to as (2004) JBiol Chem such as Alvarez.P., 279:3375-81).Enumerate method by implementing any methods availalbe in this area or this paper, be easy to measure and compare the albumen that obtains with unmodified protein and whether have long serum half-life.

Theme URP can be (a) prolongation and contains the proteic serum half-life of URP; (b) improve resultant proteic dissolubility; (c) improve protease resistant; And/or (d) reduce the resulting required any length of the proteic immunogenicity of URP that contains.Usually, theme URP contains and has an appointment 30,40,50,60,70,80,90,100,150,200,300,400 or more a plurality of aminoacid that adjoins.When mixing albumen, the URP fragmentation can be made resulting albumen comprise a plurality of URP or a plurality of URP fragment.Some or all of independent URP sequences can be shorter than 40 aminoacid, as long as the total length of proteic all the URP sequences of gained is at least 40 aminoacid.URP sequence total length in the gained protein preferably surpasses 40,50,60,70,80,90,100,150,200 or more a plurality of aminoacid.

The isoelectric point, IP of URP can be 1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,10.5,11.0,11.5,12.0,12.5 or even 13.0.

Usually, the URP sequence is rich in hydrophilic amino acid and comprises low percentile hydrophobic or aromatic amino acid.Suitable hydrophilic residue includes but not limited to glycine, serine, aspartic acid, glutamic acid, lysine, arginine and threonine.More not preferred hydrophobic residue comprises tryptophan, phenylalanine, tyrosine, leucine, isoleucine, valine and methionine when making up URP.The URP sequence can be rich in glycine, but also can be rich in glutamic acid, aspartic acid, serine, threonine, alanine or proline.Therefore main aminoacid can be G, E, D, S, T, A or P.Comprise proline residue and be tending towards reducing sensitivity proteolysis.

Comprise the hydrophilic residue and can increase URP dissolubility in water and aqueous medium under physiological condition usually.Because its aminoacid is formed, the URP sequence forms accumulative tendency in aqueous formulation low, and the fusant of URP sequence and other albumen or peptide is tending towards improving its dissolubility and reduces the tendency that forms aggregation, and this is to reduce immunogenic independent mechanism.

Design URP sequence is to avoid making albumen produce some aminoacid of undesirable property.For example, can design the URP sequence to comprise on a small quantity or not contain following aminoacid: cysteine (avoiding disulfide bond to form and oxidation), methionine (avoiding oxidation), agedoite and glutamine (avoiding deaminizating).

Rich glycine URP:

In one embodiment, theme URP comprises rich glycine sequence (GRS).For example, glycine mainly occurs, so it is the most general residue in the sequence interested.In another example, design URP sequence makes glycine residue account for about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of total amino acids at least.URP also can contain 100% glycine.In another example, URP contains at least 30% glycine, and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine is less than 20%.In another example, URP contains at least 40% glycine, and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine is less than 10%.In another example, URP contains at least 50% glycine, and the total concentration of tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine is less than 5%.

GRS length can be about 5-200 aminoacid or more a plurality of aminoacid.For example, the single GRS of adjoining can contain 5,10,15,20,25,30,35,40,45,50,55,60,70,80,90,100,120,140,160,180,200,240,280,320 or 400 or more a plurality of aminoacid.The GRS two ends all can contain glycine.

GRS also can contain other aminoacid of remarkable content, for example Ser, Thr, Ala or Pro.GRS can contain the electronegative aminoacid of remarkable content, includes but not limited to: Asp and Glu.GRS can contain the positively charged aminoacid of remarkable content, includes but not limited to: Arg or Lys.Desired is, design URP only comprises the aminoacid (being Gly or Glu) of single type, only contains the aminoacid of several types sometimes, as 2-5 seed amino acid (as being selected from G, E, D, S, T, A or P), on the contrary, general albumen and general joint are made up of the great majority of 20 seed amino acids usually.URP can comprise 30,25,20,15,12,10,9,8,7,6,5,4,3,2 or 1% amino acid position electronegative aminoacid (Asp, Glu).

Usually, the theme URP that contains GRS has about 30,40,50,60,70,80,90,100 or more a plurality of contiguous amino acid.When mixing albumen, URP can be by fragmentation, so that gained albumen comprises a plurality of URP or a plurality of URP fragment.Some or all of independent URP sequences can be shorter than 40 aminoacid, as long as the total length of proteic all the URP sequences of gained is at least 30 aminoacid.URP sequence total length in the gained protein preferably surpasses 40,50,60,70,80,90,100 or more a plurality of aminoacid.

Especially interested in the URP that contains GRS, part increases because contain the conformational freedom of glycine peptide.Denatured polypeptide in the solution has the conformational freedom of height.Described peptide makes most of conformational freedom forfeiture with combining of target spot such as receptor, antibody or protease.The forfeiture of this entropy needs that synergistic energy compensates between peptide and target spot thereof.Denatured polypeptide conformation degrees of freedom are by its aminoacid sequence decision.Compare with the peptide that the larger side chain amino acid is formed, the peptide that contains many little side chain amino acids is tending towards having higher conformational freedom.The peptide that contains glycine has king-sized degree of freedom.Expectation in solution, contain glycine peptide bond entropy than the corresponding sequence that contains alanine high 3.4 times (D ' Aquino, J.A. etc. (1996) Proteins, 25:143-56).This factor increases along with the number of glycine residue in the sequence.The result is that this type of peptide is tending towards losing more entropy in conjunction with target spot the time, has therefore weakened the ability of determining three dimensional structure with overall capacity and their employings of other albumen effect.When the Ramachandra impression (Ramachandra plots) of analyzing proteins structure, the big conformation elasticity of glycine peptide bond also clearly, the zone that the glycine peptide bond occupies is seldom by the occupied (Venkatachalam of other peptide bond, (1969) Annu Rev Biochem such as C.M., 38:45-82).Stites etc. have studied from the data base of 12,320 residues of 61 non-homogeneous high-resolution crystal structures to detect 20 aminoacid separately

, ψ conformation preference.Suppose that the distribution in the denatured state has also been reacted in observed distribution in native state albumen.Use this energy face that distributes and estimate each residue, so that calculate the relative conformational entropy of each residue with respect to glycine.Under the opposite extreme situations, replace glycine with proline, 20 ℃ of time-0.82+/-the conformation Entropy Changes of 0.08kcal/mol make compare with denatured state stablized native state (Stites, W.E. etc. (1995) Proteins, 22:132).The special role of glycine in 20 natural amino acids confirmed in these observations.

Can use natural or non-natural sequence during design motif URP.For example, table 1,2,3,4 provides the native sequences of many high glycine content.Those skilled in the art can adopt arbitrary sequence as URP or modify this sequence and reach desirable properties.When interested, preferably according to the URR that contains GRS derived from host's rich glycine sequence design in the immunogenicity of host object.The sequence preference that contains the URP of GRS has the sequence of remarkable homology from people's albumen or with the corresponding rich glycine sequence of reference man's albumen.

Table 1. contains the proteinic structural analysis of rich glycine sequence

The PDB file	Protein function	Rich glycine sequence
			1K3V	The pig parvoviral capsid	sgggggggggrgagg
1FPV	The full leukopenia syndrome virus of cat	tgsgngsgggggggsgg
			1IJS	CpV D strain, sudden change A300d	tgsgngsgggggggsgg
1MVM	Mvm (I strain) virus	ggsggggsgggg

Table 2: coding contains the open reading frame of the GRS of 300 or more glycine residues

The GRS gene

Accession number organism Gly (%) forecast function

Length by length

Arabidopsis

NP974499 64 509 579 the unknowns

(Arabidopsis?thaliana)

Bulbus Allii Cepae uncle gram bacterium

The lipoprotein that ZP_00458077 66 373 518 infers

(Burkholderia?cenocopacia)

Oryza sativa L.

XP_477841 74 371 422 the unknowns

(Oryza?sativa)

Before the cell wall of inferring

NP_910409 Oryza sativa L. 75 368 400

Body

Drosophila melanogaster

NP_610660 66 322 610 transposable elements

(Drosophila?melanogaster)

The example of table 3. people GRS

The long gene of GRS

Accession number Gly (%) hydrophobicity forecast function

Degree length

NP_000217 62 135 622 is keratin 9

NP631961 61 73 592 is TBP associated factor 15 isotypes 1

NP_476429 65 70 629 is keratin 3

Loricrin (loricrin), the cell bag

NP_000418 70 66 316 is

Film

NP_056932 60 66 638 is cytokeratins 2

The additional instance of table 4. people GRS

Aminoacid

The accession number sequence

Number

NP_006228. GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG 37

NP_787059 GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG 33

NP_009060 GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGG 32

NP_031393 GDGGGAGGGGGGGGSGGGGSGGGGGGG 27

NP_005850 GSGSGSGGGGGGGGGGGGSGGGGGG 25

NP_061856 GGGRGGRGGGRGGGGRGGGRGGG 22

NP_787059 GAGGGGGGGGGGGGGSGGGGGGGGAGAGGAGAG 33

NP_009060 GGGSGSGGAGGGSGGGSGSGGGGGGAGGGGGG 32

NP_031393 GDGGGAGGGGGGGGSGGGGSGGGGGGG 27

NP_115818 GSGGSGGSGGGPGPGPGGGGG 21

XP_376532 GEGGGGGGEGGGAGGGSG 18

NP_065104 GGGGGGGGDGGG 12

GGGS GS GGA GGGS GGGS GS GGGGGGA GGGGGGSS GGGS GTA GGHS G

The POU domain, 4 classes, transcription factor 1[homo sapiens (Homo sapiens)]

GP GGGGGP GGGGGP GGGGP GGGGGGGP GGGGGGP GGG

Contain 2 yeast domain [homo sapiens]

GGS GA GGGGGGGGGGGS GS GGGGST GGGGGTA GGG

Rich AT interactive structure territory 1B (SWI1 sample) isotype 3; The conjugated protein ELD/OSA1 of BRG1; Eld (eyelid)/Osa albumen [homo sapiens]

GA GGGGGGGGGGGGGS GGGGGGGGA GA GGA GA G

Rich AT interactive structure territory 1B (SWI1 sample) isotype 2; The conjugated protein ELD/OSA1 of BRG1; Eld (eyelid)/Osa albumen [homo sapiens]

GA GGGGGGGGGGGGGS GGGGGGGGA GA GGA GA G

Rich AT interactive structure territory 1B (SWI1 sample) isotype 1; The conjugated protein ELD/OSA1 of BRG1; Eld (eyelid)/Osa albumen [homo sapiens]

GA GGGGGGGGGGGGGS GGGGGGGGA GA GGA GA G

Rich purine element conjugated protein A; Rich purine single-stranded DNA binding protein α; Transcription activating protein PUR-α [homo sapiens]

GHP GS GS GS GGGGGGGGGGGGS GGGGGGAP GG

Regulatory factor X1; Cis regulatory factor 1; Enhancer C; MHC II class regulatory factor RFX[homo sapiens]

GGGGS GGGGGGGGGGGGGGS GST GGGGS GA G

Destructive calm territory (bromo domain) albumen [homo sapiens] that contains of leukemia

GGR GR GGR GR GSR GR GGGGTR GR GR GR GGR G

Agnoprotein [homo sapiens]

GS GGSGGS GGGP GP GP GGGGGPS GS GS GP G

Prediction: false albuminoid XP_059256[homo sapiens]

GGGGGGGGGGGR GGGGR GGGR GGGGE GGG

Zinc finger protein 28 1; ZNP-99 transcription factor [homo sapiens]

GGGGT GSS GGS GS GGGGS GGGGGGGSS G

The short isotype of rna binding protein (autoantigenicity, lethal yellow are correlated with hnRNP); Rna binding protein (autoantigenicity); Rna binding protein (autoantigenicity, lethal yellow are correlated with hnRNP) [homo sapiens]

GD GGGA GGGGGGGGS GGGGS GGGGGGG

Signal recognition particle 68kDa[homo sapiens]

GGGGGGGS GGGGGS GGGGS GGGR GA GG

KIAA0265 albumen [homo sapiens]

GGGAA GA GGGGS GA GGGS GGS GGR GT G

Zigzag congener 2; Zigzag-2[homo sapiens]

GA GGGR GGGA GGE GGAS GAE GGGGA GG

The long isotype of rna binding protein (autoantigenicity, lethal yellow are correlated with hnRNP); Rna binding protein (autoantigenicity); Rna binding protein (autoantigenicity, lethal yellow are correlated with hnRNP) [homo sapiens]

GD GGGA GGGGGGGGS GGGGS GGGGGGG

Androgen receptor; Dihydrotestosterone receptor [homo sapiens]

GGGGGGGGGGGGGGGGGGGGGGGEA G

Homology frame D11; Homology frame 4F; Hox-4.6, mice, congener; Homology frame albumen Hox-D11[homo sapiens]

GGGGGGSA GGGSS GGGP GGGGGGA GG

FZ 8; FZ (fruit bat) congener 8[homo sapiens]

GGGGGP GGGGGGGP GGGGGP GGGGG

Eye development related gene [homo sapiens]

GR GGA GS GGA GS GAA GGT GSS GGGG

Homology frame B3; Homology frame 2G; Homology frame albumen Hox-B3[homo sapiens]

GGGGGGGGGGGS GGS GGGGGGGGGG

Chromosome 2 open reading frame 29[homo sapiens]

GGS GGGR GGAS GP GS GS GGP GGPA G

DKFZP564F0522 albumen [homo sapiens]

GGHH GDR GGGR GGR GGR GGR GGRA G

Prediction: skip even (even-skipped) homologous protein 2 (EVX-2) similar [homo sapiens] to the homology frame

GSR GGGGGGGGGGGGGGGGA GA GGG

Ras homologous genes family, U member; Ryu GTP enzyme; The reactive Cdc42 congener of Wnt-1; 2310026M05Rik; Gtp binding protein 1; CDC42 sample GTP enzyme [homo sapiens]

GGR GGR GP GEP GGR GRA GGAE GR G

Scratching (scratch) 2 albumen; Transcribe and suppress son scratching 2; Scratching (fruit bat congener) 2, zinc finger protein [homo sapiens]

GGGGGDA GGS GDA GGA GGRA GRA G

The A of p120 family, the member 1; GAR1 albumen [homo sapiens]

GGGR GGR GGGR GGGGR GGGR GGG

Keratin 1; Keratin-1; Cytokeratin 1; Hair α albumen [homo sapiens]

GGS GGGGGGSS GGR GS GGGSS GG

False albuminoid FLJ31413[homo sapiens]

GS GP GT GGGGS GS GGGGGGS GGG

Singly cut (one cut) domain, the family member 2; Singly cut 2[homo sapiens]

GAR GGGS GGGGGGGGGGGGGGP G

The POU domain, 3 classes, transcription factor 2[homo sapiens]

GGGGGGGGGGGGGGGGGGGGGD G

Prediction: to (REF1-I) (alliance of AML-1 and LEF-1) (Aly/REF) similar [homo sapiens] of THO complex subunit 4 (Tho4) (RNA and export factor bindin 1)

GGTR GGTR GGTR GGDR GR GR GA G

Prediction: with (REF1-I) (alliance of AML-1 and LEF-1) (Aly/REF) [homo sapiens] of THO complex subunit 4 (Tho4) (RNA and output factor bindin 1)

GGTR GGTR GGTR GGDR GR GR GA G

The POU domain, 3 classes, transcription factor 3[homo sapiens]

GA GGGGGGGGGGGGGGA GGGGGG

The A of p120 family, the member 1; GAR1 albumen [homo sapiens]

GGGR GGR GGGR GGGGR GGGR GGG

Fibrillarin; 34-kD kernel scleroderma antigen; RNA, U3 small nut core interacting protein 1[homo sapiens]

GR GR GGGGGGGGGGGGGR GGGG

Zinc finger protein 579[homo sapiens]

GR GR GR GR GR GR GR GR GR GGA G

Calpain, small subunit 1; Calpain; Calpain, little polypeptide; Calpain 4, small subunit (30K); Ca-dependent protease, small subunit [homo sapiens]

GA GGGGGGGGGGGGGGGGGGGG

Keratin 9[homo sapiens]

GGGS GGGHS GGS GGGHS GGS GG

Jaw (forkhead) frame D1; The jaw frame activator 4 of being correlated with; Jaw albumen, fruit bat, congener sample 8; Jaw albumen (fruit bat) sample 8[homo sapiens]

GA GA GGGGGGGGA GGGGSA GS G

Prediction: to RIKEN cDNA C230094B15 similar [homo sapiens]

GGP GT GS GGGGA GT GGGA GGP G

GGGGGGGGGA GGA GGA GSA GGG

Cadherin 22 precursors; P of Rats B-cadherin is directly to congener [homo sapiens]

GGD GGGSA GGGA GGGS GGGA G

AT is in conjunction with transcription factor 1; AT motif binding factor 1[homo sapiens]

GGGGGGS GGGGGGGGGGGGGG

Take off middle embryo protein; The t box, brain, 2; Take off middle embryo protein (Africa xenopus (Xenopus laevis)) congener [homo sapiens]

GP GA GA GS GA GGSS GGGGGP G

Phosphatidylinositol transfer protein, film is in conjunction with 2; PYK2N end structure territory-interaction receptor 3; Retinal degeneration B α 2 (fruit bat) [homo sapiens]

GGGGGGGGGGGSS GGGGSS GG

Sperm related antigen 8 isotypes 2; Sperm membrane albumen 1[homo sapiens]

GS GS GP GP GS GP GS GP GH GS G

Prediction: RNA binding motif albumen 27[homo sapiens]

GP GP GP GP GP GP GP GP GP GP G

Conjugated protein 1 isotype 1 of AP1 γ subunit; γ-synergin; The conjugated protein 1[homo sapiens of adapter associated protein complex 1 γ subunit]

GA GS GGGGAA GA GA GSA GGGG

Conjugated protein 1 isotype 2 of AP1 γ subunit; γ-synergin; The conjugated protein 1[homo sapiens of adapter associated protein complex 1 γ subunit]

GA GS GGGGAA GA GA GSA GGGG

Comprise 1 anchorin repetition and barren (sterile) α motif domain; Comprise 1 anchorin repetition and SAM domain [homo sapiens]

GGGGGGGS GGGGGGS GGGGGG

Methyl-CpG binding domain protein 2 isotype 1[homo sapiens]

GR GR GR GR GR GR GR GR GR GR G

Three functional domains (PTPRF interaction) [homo sapiens]

GGGGGGGS GGS GGGGGS GGGG

Jaw albumen box D3[homo sapiens]

GGEE GGAS GGGP GA GS GSA GG

Sperm related antigen 8 isotypes 1; Sperm membrane albumen 1[homo sapiens]

GS GS GP GP GS GP GS GP GH GS G

Methyl-CpG binding domain protein matter 2 testes specificity isotypes [homo sapiens]

GR GR GR GR GR GR GR GR GR GR G

Cell death is regulated hole (aven); Programmed cell death 12[homo sapiens]

GGGGGGGGD GGGRR GR GR GR G

Nonsense transcript regulon 1; The δ unwindase; Frameshift mutation 1 congener (saccharomyces cerevisiae (S.cerevisiae)) makes progress; Nonsense mRNA reduces the factor 1; Yeast Upflp congener [homo sapiens]

GGP GGP GGGGA GGP GGA GA G

Small-conductance calcium-activated potassium channel protein 2 isotype a; The apamin responsive type activated potassium channel of little electric conductance Ca2+ [homo sapiens]

GT GGGGST GGGGGGGGS GH G

SRY (sex-determining region Y)-box 1; SRY dependency HMG-box gene 1[homo sapiens]

GPA GA GGGGGGGGGGGGGGG

Transcription factor 20 isotypes 2; Stromelysin-1 platelet derived growth factor response element is conjugated protein; Stromelysin 1PDGF response element is conjugated protein; SPRE is conjugated protein; Nuclear factor SPBP[homo sapiens]

GGT GGSS GSS GS GS GGGRR G

Transcription factor 20 isotypes 1; Stromelysin-1 platelet derived growth factor response element is conjugated protein; Stromelysin 1PDGF response element is conjugated protein; SPRE is conjugated protein; Nuclear factor SPBP[homo sapiens]

GGT GGSS GSS GS GS GGGRR G

Ras interaction protein 1[homo sapiens]

GS GT GTT GSS GA GGP GTP GG

BMP-2 induction type kinases isotype b[homo sapiens]

GGS GGGAA GGGA GGA GA GA G

BMP-2 induction type kinases isotype a[homo sapiens]

GGS GGGAA GGGA GGA GA GA G

Jaw albumen box C1; The jaw albumen activator 3 of being correlated with; Jaw albumen, fruit bat, congener sample 7; Jaw albumen (fruit bat) sample 7; Aplasia of iris (iridogoniodysgenesis) 1 type [homo sapiens]

GSS GGGGGGA GAA GGA GGA G

Splicing factor p54; Rich arginic 54kDa nucleoprotein [homo sapiens]

GP GPS GGP GGGGGGGGGGGG

V-maf muscular aponeurotic fibrosarcoma oncogene congener; Fowl muscular aponeurotic fibrosarcoma (MAF) proto-oncogene; V-maf muscular aponeurotic fibrosarcoma (birds) oncogene congener [homo sapiens]

GGGGGGGGGGGGGGAA GA GG

Small nut nucleoprotein D1 polypeptide 16kDa; SnRNP core protein D1; The Sm-D autoantigen; Small nut nucleoprotein D1 polypeptide (16kD) [homo sapiens]

GR GR GR GR GR GR GR GR GR GG

False albuminoid H41[homo sapiens]

GSA GGSS GAA GAA GGGA GA G

The URP (NGR) that contains non-glycine residue:

Select non-glycine sequence among these HRS to optimize URP and to contain the proteic character of required URP.For example, can optimize the URP sequence to improve resultant albumen for particular organization, particular cell types or cytophyletic selectivity.For example, can mix not is wide expression but at one or more bodily tissues with suffer from the protein sequence of differential expression in the selected tissue of disease, these bodily tissues comprise heart, liver, prostate, lung, kidney, bone marrow, blood, skin, bladder, brain, muscle, nerve and selected tissue, and these diseases comprise infectious disease, autoimmune disease, nephropathy, neuropathy, heart disease and cancer etc.Can use the sequence of the specific growth origin of representative, as the sequence of when ectoderm, entoderm and the mesoderm of embryo, adult form, expressing in the multicellular organisms.Also can utilize to relate to the particular biological process, include but not limited to the sequence that Cycle Regulation, cell differentiation, apoptosis, chemotactic, cell movement and cytoskeleton are reset.Also can utilize the protein sequence of other non-wide expression to guide resulting albumen to arrive specific subcellular location: extracellular matrix, nucleus, kytoplasm, cytoskeleton, endochylema and/or intracellular membrane structure include but not limited to by alveole, Golgi body, endoplasmic reticulum, endosome, lysosome and mitochondrion.

Known multiple tissue specificity, cell type specificity, Subcellular Localization specific sequence also can obtain from several albumen databases.By produce at random or partly at random URP sequence library, it is injected into animal or patient and detects the sequence that has required tissue selectivity in the tissue samples and obtain selectivity URP sequence.Available Mass Spectrometer Method sequence.Use that similar approach can be selected to be beneficial in oral, buccal, intestinal, nasal cavity, the sheath, the URP sequence of peritoneum, rectum or skin picked-up.

Outstanding interested is the URP sequence that is rich in positively charged arginine or lysine zone relatively that contains that helps cellular uptake or wear the film transhipment.Can design the URP sequence makes it contain the responsive sequence of one or more protease.In case product of the present invention arrives its target position, can cut away this URP sequence.This cutting can trigger the usefulness (prodrug activation) of pharmaceutical active domain or strengthen combining of cleaved products and receptor.Can design the URP sequence makes it be with too much negative charge by introducing aspartic acid or glutaminic acid residue.Cherish a special interest be contain more than 5%, more than 6%, 7%, 8%, 9%, 10%, 15%, 30% or more glutamic acid and be less than 2% lysine or arginic URP.This type of URP carries too much negative charge, because the Coulomb repulsion effect between the single negative charge of this peptide makes it be tending towards taking open conformation.The too much negative charge of this kind causes that its hydrodynamic radius effectively increases and therefore reduce the kidney clearance rate of this quasi-molecule.Therefore, can be by electronegative amino acid whose frequency in the control URP sequence and the effective net charge and the hydrodynamic radius that distribute and reconcile the URP sequence.Too much negative charge is with on human or animal's majority tissue and surface.By the URP of design with too much negative charge, can with contain the gained albumen of URP and different surfaces such as blood vessel, health tissues or not the non-specific interaction between the isoacceptor reduce to minimum.

URP can contain (motif) _xThe repetition aminoacid sequence of form, wherein sequence motifs forms direct repetition (being ABCABCABCABC) or oppositely repeats (ABCCBAABCCBA), and these multiple number of times can be 2,3,4,5,6,7,8,9,10,12,14,16,18,20,22,24,26,28,30,35,40,50 or more.The repetition of URP or URP inside often only comprises 1,2,3,4,5 or 6 kind of dissimilar aminoacid.URP usually by length be 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,22,24,26,28,30,32,34,36 or more a plurality of amino acid whose human amino acid sequence repeat to form, but URP can be that 20,22,24,26,28,30,32,34,36,3840,42,44,46,48,50 amino acid whose inhuman aminoacid sequences are formed by length also.

The URP of derived from human sequence:

But URP derived from human sequence.The people's gene group comprises the subsequence that much is rich in specific amino acids.What cherish a special interest is this type of aminoacid sequence that is rich in hydrophilic amino acid such as serine, threonine, glutamic acid, aspartic acid or glycine.What cherish a special interest is this type of subsequence that contains hydrophobic amino acid hardly.Estimate that this type of subsequence is a destructuring and solvable at the aqueous solution camber.Can modify this type of people's subsequence with its practicality of further improvement.Figure 17 shows the exemplary human sequence of rich serine, and is separable as theme URP.Exemplary DSPP contains 670 amino acid whose subsequences, and wherein 64% residue is a serine, and most other position is hydrophilic amino acid, as aspartic acid, agedoite and glutamic acid.Therefore this sequence repeats extremely that information content is low.Can directly utilize the proteic subsequence of this type of people.Desired is, to keep its general nature but make its mode that is more suitable for medicinal application modify this sequence.The sequence example relevant with DSPP is (SSD) _n, (SSDSSN) _n, (SSE) _n, wherein n is about 4-200.

When the URP that designer's individual immunity originality reduces, especially wish to use from the proteic sequence of people.Initiation is the described proteic fragments of peptides of MHC 2 receptoroid submissions for the committed step of foreign protein immunne response.These MHC II binding fragments can be detected by TXi Baoshouti, trigger the propagation of t helper cell and start immunne response.From pharmaceutical protein reduce t cell epitope be considered to reduce the method that causes the immunne response risk (Stickler, M. etc. (2003) J Immunol Methods, 281:95-108).MHC II receptor interacts with the epi-position that has as 9 amino acid whose regional displayed polypeptides usually.Therefore, if all or most 9 possible aggressiveness subsequences can find from people's albumen, the repeating of these sequences and sequence can not taken as exogenous array by patient, just can reduce the risk that causes in the patient body proteic immunne response.Have the human sequence that suitable aminoacid is formed by oligomerization or connection, the human sequence can be mixed the URP design.They can be direct repetition or oppositely repeat or different multiple mixing.For example can be with the oligomerization of sequence shown in the table 2.This type of oligomer has low immunogen risk.Yet the catenation sequence of monomeric unit still can comprise the immunoreactive t cell epitope of triggering, as shown in Figure 3.Can further reduce the risk of initiation immunne response based on multiple overlapping human sequence's URP by design.This method as shown in Figure 4.Among Fig. 2 the URP sequence be designed to oligomer based on multiple human sequence design, so each 9 aggressiveness subsequence can find in people's albumen in the oligomer.In these designs, each 9 aggressiveness subsequence is the human sequence.Fig. 5 shows the example based on three human sequences' URP.Also can design the URP sequence, so all possible 9 aggressiveness subsequences appear at all in same people's albumen in the oligomerization URP sequence based on single human sequence.Fig. 6 has shown the example based on the POU domain of rich glycine and proline.Fig. 6 shows in the URP sequence repeated monomer proteic fragment of only behaving, and its side joint sequence is identical with recurring unit.Can design non-oligomerization URP sequence according to people's albumen equally.The most important condition is that all 9 aggressiveness subsequences can find in the human sequence.The aminoacid of sequence is formed and is preferably contained hydrophobic residue hardly.What cherish a special interest is based on URP sequence human sequence design and that comprise a large amount of glycine residues.

Utilize this or similar scheme, can design and comprise a class URP who host interested is had the reduced immunogenicity repetitive sequence.Interested host can be any animal, comprises vertebrates and invertebrates.Preferred host is a mammal, as primates (as chimpanzee and people), cetacean (as whale and dolphin), chiropterans (as Vespertilio), Perissodactyla (as horse and rhinoceros), Rodents (as rat) and some insectivora such as suslik, mole and Rrinaceus earopaeus.When selecting the people as the host, URP contains the repetitive sequence or the unit of a plurality of copies usually, and the section majority that wherein comprises about 6-15 contiguous amino acid comes across in one or more natural human albumen.Also can design the section majority that comprises about 9-15 contiguous amino acid comes across in one or more natural human albumen.The employed most of section of this paper refers to more than about 50%, preferred 60%, preferred 70%, preferred 80%, preferred 90%, preferred 100% section.Each about 6-15 aminoacid in the expectation repetitive, preferably about 9-15 is amino acid whose may all to be appeared in one or more natural human albumen by section.URP can comprise a plurality of repetitives or sequence, for example has 2,3,4,5,6,7,8,9,10 more a plurality of repetitives alive.

Substantially do not contain the URP design of human T-cell's epi-position:

Can design the URP sequence makes it not contain the epi-position that can be discerned by the human T-cell substantially.For example, can synthesize a series of half random sequences that the aminoacid that contains the destructuring conformation that helps to form degeneration is formed, and estimate whether have in these sequences whether human T-cell's epi-position and they are the human sequence.Described the experiment that is used for human T-cell's epi-position (Stickler, M. etc. (2003) J Immunol Methods, 281:95-108).Cherish a special interest be can be under the situation that does not produce t cell epitope and non-human sequence the peptide sequence of oligomerization.Whether have t cell epitope and inhuman 6-15 aggressiveness by detecting these sequences in directly repeating, particularly 9 aggressiveness subsequences are realized above-mentioned purpose.Another kind method is to utilize aforementioned human sequence to assemble a plurality of peptide sequences that chapters and sections are assembled into repetitive.Another alternative be the design URP sequence of utilizing the low scoring of epi-position prediction algorithm such as T epi-position (TEPITOPE) (Sturniolo, T. etc. (1999) Nat Biotechnol, 17:555-61).Another method of avoiding t cell epitope is to avoid being used as in the middle of the peptide displaying on MHC the aminoacid of anchor residues, as M, I, L, V, F.Hydrophobic amino acid and positively charged aminoacid often can be used as this type of anchor residues, at utmost reduce its frequency in the URP sequence and reduce chance and the therefore immunoreation of initiation that t cell epitope produces.Common selected URP comprises the subsequence that finds at least in a kind of people's albumen, and comprises the hydrophobic amino acid of lower content.

Design URP sequence can be by avoiding reaching this purpose with the repeatability that at utmost reduces coding DNA to optimize protein production.URP sequence such as polyglycine can have very excellent drug characteristic, but the reiterated DNA sequences that high GC content and existing can cause recombinating in the DNA sequence of GRS of encoding causes the difficulty of manufacturing.

As mentioned above, can design in the multiple URP sequence of amino acid levels height.Therefore the URP sequence has very low information content and can reduce and cause immunoreactive risk.

Comprising the non-limitative example that repeats amino acid whose URP is: polyglycine, polyglutamic acid, poly aspartic acid, Poly(Ser) L-Serine polymer, poly threonine, (GX) _n, wherein G is that glycine and X are serine, aspartic acid, glutamic acid, threonine or proline, n is at least 20; (GGX) _n, wherein X is serine, aspartic acid, glutamic acid, threonine or proline, n is at least 13; (GGGX) _n, wherein X is serine, aspartic acid, glutamic acid, threonine or proline, n is at least 10; (GGGGX) _n, wherein X is serine, aspartic acid, glutamic acid, threonine or proline, n is at least 8; (G _zX) _n, wherein X is serine, aspartic acid, glutamic acid, threonine or proline, n is at least 15, and z is 1-20.

These multiple numbers can be the Any Digit of 10-100.Product of the present invention can comprise the URP sequence of half random sequence.Example can be half random sequence that comprises at least 30,40,50,60 or 70% glycine, and wherein the combination total concentration of glycine good dispersion and tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine is less than 70,60,50,40,30,20 or 10%.Preferred partly at random the URP sequence total concentration that comprises at least 40% glycine and tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine be less than 10%.The preferred sequence of URP at random comprises the total concentration of at least 50% glycine and tryptophan, phenylalanine, tyrosine, valine, leucine and isoleucine less than 5%.Can design the URP sequence by two or more short URP sequences or URP sequence fragment are merged.This merging can better be regulated the medicinal property that contains URP sequence product and reduce the repeatability of the DNA sequence of coding URP sequence, thereby improves the expression of URP coded sequence and reduce its reorganization.

Design also selects to have the URP sequence of following desired characteristic: a) the height hereditary stability of coded sequence in producing the host; B) expression height; C) low (prediction/calculate) immunogenicity; D) in serum albumin enzyme and/or other protease, has high stability; E) hydrodynamic radius is big under the physiological condition.The exemplary method that acquisition meets the URP sequence of a plurality of standards is to make up the library of candidate sequence and identify suitable subsequence from the library.The library can comprise at random and/or semirandom sequence.Useful especially is the codon library, and this is the dna molecular library that comprises the multiple codon of same amino acid residue.The sub-randomization of applied cryptography is selected certain type or most or all amino acid positions.The real password sublibrary single amino acid sequence of only encoding, but be easy to and the aminoacid library merges, be formed on the dna molecular colony of same residue position encoded (relevant or irrelevant) ispol.Can identify in the dna level low repeatability but the gene of coding high duplication aminoacid sequence with the codon library.This point is very useful, because reiterated DNA sequences is tending towards reorganization, causes unstability.Also can make up the multifarious codon of the limited aminoacid of coding library.This type of library allows to introduce in some position of sequence the aminoacid of limited quantity, yet other position allows codon to change the same monoamino-acid but all codons are encoded.Can be when oligonucleotide is synthetic by mix mixture of ribonucleotides composite part random oligonucleotide at same position.This type of part random oligonucleotide can merge by overlapping PCR or based on the method that connects.Specifically, half random oligonucleotide of rich glycine sequence but multimerization is encoded.These oligonucleotide are different on length, sequence and codon use.The result is to obtain the library of candidate URP sequence.Another method that produces the library is again with described sequence incomplete randomization behind the synthetic homing sequence.Gene that can be by in mutant, cultivating coding URP sequence or by amplification coding gene in the mutation environment realize (Leung, D. etc. (1989) Technique, 1:11-15).Utilize several methods from the library, to identify URP sequence with required character.In producing the host, cultivate the sequence that this library enrichment has the height hereditary stability.Unsettled sequence can totally be suddenlyd change, and can identify by dna sequencing.Can use the method screening known to the those skilled in the art or select, but to identify the URP sequence variants of high level expression.For example can be from cultivating a plurality of libraries separator and comparing its expression.By gel analysis, analysis chromatograph or multiple method mensuration expression based on ELISA.With candidate URP sequence library and the expression that helps measuring every kind of sequence variants such as the sequence label fusion of myc label, His label, HA label.Other method is that library and enzyme or other reporter proteins such as green fluorescent protein are merged.What cherish a special interest is with library and selected marker such as beta-lactamase or kanamycin-acyltransferase fusion.Can utilize antibiotic to select enrichment expression height and the good variant of hereditary stability.Hatch back screening complete sequence has good protease resistant with evaluation variant with protease.The approach of effectively identifying protease resistant URP sequence is phage display or relevant methods of exhibiting.The multiple systems that the sequence of quick proteolysis can take place by the phage display enrichment has been described.Adopt these methods to come rich protein enzyme resistance sequence easily.For example, can between affinity tag and M13 phage pIII albumen, clone candidate URP sequence library.Make this library contacting protease then or contain biological sample such as the blood or the lysosome prepared product of protease.Can catch the phage that contains the protease resistant sequence by combining after the protease effect with affinity tag.What cherish a special interest is the sequence that can resist the degraded of lysosome prepared product, because the lysosome degraded is dendritic cell and the committed step of other antigen presenting cells in antigen presentation.Can use phage display to identify not and the bonded candidate URP of specific immune serum sequence to have the URP sequence of reduced immunogenicity with evaluation.Can utilize candidate URP sequence or URP sequence library immune animal to produce the antibody of URP sequence in the anti-library.Gained serum is used for the phage elutriation to remove or to identify by the sequence of gained immune serum antibody recognition.Also can utilize the URP sequence variants that other method such as antibacterial are showed, yeast is showed, ribosomal display identification has desirable characteristics.Other method is to utilize mass spectrum to identify interested URP sequence.For example, with candidate URP sequence library and protein of interest enzyme or biological sample is hatched and utilize mass spectrum to identify degradation-resistant sequence.Can utilize similar methods to identify the URP sequence that is beneficial to orally ingestible.Can be with the mixture feeding animals or the people of candidate URP sequence, and utilize mass spectrum to identify to stride and organize barrier (being skin etc.) transhipment or the highest variant of ingestion efficiency.In a similar manner, identify and to be beneficial to other picked-up mechanism as through the URP of lung, intranasal, rectum, transdermal delivery sequence.Also can identify the URP sequence of the URP sequence that is beneficial to cellular uptake or anti-cell picked-up.

Can design the URP sequence by any URP sequence of merging said method design or the fragment of URP sequence.In addition, can use half random method is optimized the sequence based on above-mentioned Rule Design.What cherish a special interest is carry out for improving the purpose that strengthens proteic expression and the hereditary stability of raising encoding gene in producing the host codon optimized.For being rich in the very high URP sequence of glycine or aminoacid sequence repeatability, codon optimized extremely important.Can utilize computer program to carry out that codon optimized (22:346-53), some of them can at utmost reduce intermittently (Coda Genomics Inc.) (examining the genomics company that reaches) of ribosome for Gustafsson, C. etc. (2004) Trends Biotechnol.When design URP sequence, can consider some character.Can at utmost reduce the repeatability of DNA sequences encoding.In addition, can avoid or at utmost reduce to use produce the codon of seldom using among the host (being a leucine codon in AGG and AGA arginine codon and the escherichia coli (E.coli)).DNA sequence with high-level glycine is tending towards high GC content, can cause unstability and expression low.The preferential GC content of selecting to make the URP coded sequence is suitable for making the organic codon of production of URP when therefore, possible.

Can process by the complete synthesis or synthetic enzyme that adds, clone, PCR and overlapping extension as restriction enzyme-mediated utilize a step or multistep to make the URP encoding gene.Making up the URP module makes URP module coding gene have low repeatability and amino acid sequence coded has highly repeatability.Figure 11 shows this method.The first step makes up short relatively URP sequence library.This can be the pure cipher sublibrary, and each library member has identical aminoacid sequence but has the different coded sequence of many kinds.The library member who helps identifying good representation is merged in this library and reporter protein.The example of suitable reporter gene has green fluorescent protein, luciferase, alkali phosphatase, beta galactosidase.Can identify by screening can be in the short URP sequence of selected host's organism middle and high concentration expression.Then, generate at random URP dimer library and repeat to screen high level expression.Extension or similar clone technology are carried out dimerization by connecting, overlapping.Can repeat several dimerization process and follow-up screening and reach Len req until gained URP sequence.Alternatively, contain the separator of non-required sequence with elimination to cloning to check order in the library.The initial library of short URP sequence allows aminoacid sequence to change.For example, can make some hydrophilic amino acids appear at described position some codon randomizations.In the process of multimerization repeatedly, except that high level expression is screened, also can screen, as dissolubility, protease resistant to other characteristic of member in the library.Except dimerization URP sequence, also can produce longer polymer.This can increase the length of URP module faster.

Many URP sequences are rich in specific amino acids.Because encoding gene may contain the repetitive sequence that is subject to recombinate, therefore utilize recombinant technique to be difficult to produce this type of sequence.And, because the tRNA that loads separately in producing the host is limited, so but the gene limiting expression of specific cryptosystem appears in high frequency.An example is the recombinant production of GRS.4 kind of three disjunctor coding glycine residue, they are: GGG, GGC, GGA and GGT.The result is that the gene of coding GRS is tending towards having high GC content and repeatability is high especially.The codon preference of producing the host is another challenge.When (produce host be) escherichia coli, two codon glycine GGA and GGG seldom are used to high expressed protein.Therefore the gene that is starved of coding URP sequence carries out codon optimized.Can use the optimization for program codon of the close production of consideration host numeral preference to use (Gustafsson, C. etc. (2004) Trends Biotechnol, 22:346-53).In addition, can make up the codon library, encode same aminoacid sequence but use different codons of its all members.Can screen the member that this type of library obtains high expressed and inheritance stability, they are particularly suitable for large-scale production and contain the URP product.

Multivalence destructuring recombiant protein (MURP):

As mentioned above, theme URP is very useful module when design has the albumen of therapeutic value.Therefore, the invention provides the albumen that comprises one or more theme URP.This albuminoid called after destructuring recombiant protein (MURP) herein.

In order to make up MURP, one or more URP sequences and protein N terminal or C-terminal are merged, or be inserted in the middle of the albumen, as be inserted in the albumen ring or between the proteins of interest module, make the gained modified protein compare character with unmodified protein and improve.The total length that is connected in proteic URP sequence can be 40,50,60,70,80,90,100,150,200 or more a plurality of aminoacid.

Theme MURP has the character of one or more improvement as detailed below.

The half-life of improving:

The URP sequence is added in pharmaceutically active protein can improves this proteic multiple character.Specifically, adding long URP sequence can the proteic serum half-life of significant prolongation.This type of URP comprises usually at least about 40,50,60,70,80,90,100,150,200 or more a plurality of amino acid whose aminoacid sequence.

Can make the URP fragmentation, so that gained albumen comprises a plurality of URP or a plurality of URP fragment.Some or all of these independent URP sequences can be shorter than 40 aminoacid, as long as the total length of all URP sequences is at least 30 aminoacid in the gained albumen.Preferably, the URP total length surpasses 40,50,60,70,80,90,100,150,200 or more a plurality of aminoacid in the gained albumen.In one aspect, fusion URP can increase proteic hydrodynamic radius and so reduce kidney and remove this proteic speed from blood.Can utilize ultracentrifugation, size exclusion chromatograph or light scattering to detect of the increase of gained fusion rotein hydrodynamic radius with respect to unmodified protein.

The tissue selectivity that improves:

The hydrodynamic radius increase causes that the tissue infiltration reduces, and can utilize the side effect of this pharmaceutically active protein of naming a person for a particular job to reduce to minimum.On the books, because enhanced property and delay (EPR) effect, hydrophilic polymer is tending towards selectivity and accumulates in tumor tissues.The potential cause of EPR effect be tumor vascular seepage attribute (McDonald, D.M. etc. (2002) Cancer Res, 62:5381-5) and lack lymph in the tumor tissues and flow out.Therefore, can strengthen the selectivity of the pharmaceutically active protein that is used for tumor tissues by the adding hydrophilic polymer.Equally, mix the therapeutic index that theme URP can improve given pharmaceutically active protein.

Degraded protection and immunogenicity reduce:

Add the URP sequence and can significantly improve proteinic protease resistant.Can design the URP sequence and make and himself have protease resistant, and this albumen be avoided near digestive enzyme by it is connected with albumen.The URP sequence can be added pharmaceutically active protein to reach the bad interactional purpose that reduces albumen and other receptor or surface.In order to reach this purpose, the URP sequence should be joined in the pharmaceutically active protein with the approaching protein loci that causes this badness contact.Specifically, the URP sequence can be joined pharmaceutically active protein to reduce itself and immune any component interaction to stop immunne response to product of the present invention.The URP sequence is joined the interaction of pharmaceutically active protein energy minimizing and preexisting antibody or B-cell receptor.And, add the URP sequence and can reduce picked-up and the processing of antigen presenting cell product of the present invention.Preferably add one or more URP sequences to reduce its immunogenicity in albumen, because it can suppress immunne response in a lot of species, this makes people to predict the immunogenicity of product in patient according to animal data.Separately its immunogenic these type of species of test can not be used for identifying or removing or carry out sequence method relatively with the human sequence based on human T-cell's epi-position.

Interrupt t cell epitope:

The URP sequence is introduced albumen to interrupt t cell epitope.Very useful for this point of the albumen that merges a plurality of separation function modules.The formation of t cell epitope needs the peptide section of proteantigen to combine with MHC.Being generally 9 short aminoacid sections that adjoin residue in MHC molecule and the submission peptide interacts.The direct fusion of different binding modules can cause two adjacent structure territories of t cell epitope leap in the protein molecular.Utilize URP module separation function module to stop the formation of this generic module great-leap-forward t cell epitope as shown in Figure 7.Between functional module, insert the URP sequence and also can disturb the proteolysis in the antigen presenting cell to process, cause immunogenic further reduction.Another method that reduces the immunogenicity risk is destroyed the t cell epitope in the product functional module.When being microprotein, a kind of method is to make ring (not participating in targeted integration) rich glycineization between some cysteine.In structure, contain in the microprotein of minority cysteine, in fact most or all residues that do not participate in targeted integration can be replaced with glycine, serine, glutamic acid and threonine, thereby under the prerequisite that does not influence the target spot affinity, reduce immunogenic possibility.For example, can be undertaken by all residues being carried out " glycine scanning ", each residue is replaced by glycine in this process, utilizes phage display or screening to select the clone who keeps targeted integration then, and merges the glycine replacement of all permissions.Usually, comparing functional module with the URP module, to comprise the probability of t cell epitope much higher.Reduce the frequency that t cell epitope occurs in the functional module by utilizing little hydrophilic residue such as gly, ser, ala, glu, asp, asn, gln, thr to replace all perhaps how non-key amino acid residues.Utilize some can identify the position that allows replacement in the functional module at random or based on the protein engineering method of structure.

The dissolubility that improves:

The protein function module has limited dissolubility.Specifically, binding modules is tending towards carrying hydrophobic residue on the surface, thereby limits its dissolubility and cause gathering.Utilize the URP sequence to separate or this type of functional module of side joint can improve the overall solubility of products therefrom.URP module for hydrophilic that carries remarkable ratio or charged residue is especially true.By can reduce the intramolecular interaction of these functional modules with solubility URP module separation function module.

The pH character of improving and the homogeneity of product electric charge:

Design URP sequence makes it carry excessive negative charge or positive charge.The result is that they produce electrostatic field to fusion partners, can be used for changing the pH character of enzyme or binding interactions.And the electrostatic field of charged URP sequence can increase the homogeneity of protein product surface charge pKa value, and this will cause sharpening (sharpen) and the isoelectrofocusing or the isolating sharpening of chromatofocusing of the pH character of ligand interaction.

Because product pKa sharp-pointed (sharp) causes the purification character of improvement:

Every seed amino acid itself in the solution has single fixed pKa, and this is that half functional group is by protonated pH value.Typical albumen has polytype residue, because close mutually and proteic breathing effect, they change effective pKa each other in many ways.Therefore, under the pH of wide region condition, general albumen can take to have separately the hundreds of different ionized form of different molecular weight and net charge, because there is the combination of a variety of charged and neutral amino acid residues.This is known as wide ionizing spectrum and such proteic analysis (being mass spectrum) and purification is caused difficulty.

PEG is not charged and do not influence the protonated spectrum of connected protein, makes it keep wide protonated spectrum.Yet the URP that contains high-load Gly and Glu in principle only exists with two states: be neutrality (COOH), electronegative (COO when pH is higher than glutamic acid pKa when pH is lower than glutamic acid pKa ^-).The URP module can form single, even Ionized molecule type and produce single quality in mass spectrum.

Required is, MURP and single charge type (Glu) are distributed in URP amalgamation and expression on the whole URP module with constant interval.Can be chosen in and mix 25-50 Glu residue among every 20kD URP, and all this 25-50 residues have very similar pKa.

In addition, with 25-50 electronegative being added to, make the pKa of isoelectric point, IP and free glutamic acid very close such as electric charge homogeneity and its isoelectric point, IP of sharpening that can increase product in IFN, hGH or the GCSF small proteins such as (only containing 20 charged residues).

Compare with traditional PEGization, the raising of albumen colony electric charge homogeneity can bring favourable processing characteristics, as the characteristic in ion exchange, isoelectrofocusing, mass spectrum etc.

The preparation that improves and/or send:

Adding URP can significantly simplify the preparation of products therefrom and/or send in pharmaceutically active protein.Design URP sequence makes its hydrophilic very high, thereby improves people's proteic (for example) dissolubility, and these people's albumen often contain and are used for and other protein bound hydrophobic speckle (patch).The preparation of this type of people's albumen such as antibody is had a challenge very much, often limits its concentration and sends selection.URP can reduce the precipitation and the gathering of product, and can use the better simply preparation that comprises less composition, and these compositions stable product in solution is required usually.Contain the deliquescent raising of URP sequence product and make it therefore reduce the volume injected of injectable product, can only limit to family's injection of very small size injection like this with the higher concentration preparation.Add the URP sequence and also can simplify the storage of gained preparation product.Can add in pharmaceutically active protein that URP is beneficial to that it is oral, pulmonary, rectum or intranasal picked-up.Because allow higher production concentration and the stability that has strengthened product, so the URP sequence can be beneficial to multiple delivery modality.The URP sequence that design is beneficial to membrane permeation can reach further improved purpose.

Improve and produce:

Adding URP sequence has remarkable benefit to the production of products therefrom.Many recombinant products, especially natural human albumen, being tending towards in process of production forming cannot or hardly dissolved aggregation, even can occur once more after it is removed from end product.These (natural human) albumen of this Chang Yinwei contact with other (natural human) albumen by hydrophobic speckle, and for immunogenic consideration, these residues that suddenly change are risky.Yet URP can increase this type of proteic hydrophilic, and improves its preparation under the prerequisite that need not the mutant human protein sequence.The URP sequence can be beneficial to protein folding to reach its native state.The many pharmaceutically active proteins that utilize recombinant technique to produce are non-natural state of aggregations.Need carry out degeneration to these products, hatch under its condition that is folded into the natural activity state allowing then.The side reaction that takes place in the renaturation process of being everlasting is to produce aggregation.URP sequence and proteic fusions significantly reduce its tendency that forms aggregation, therefore are beneficial to the folding of product Chinese materia medica active component.Compare with polymer-modified albumen, the product that contains URP is easier to preparation.After the purification activated protein, the chemical polymerization thing is modified needs extra modification and purification step.On the contrary, the URP sequence can be made with pharmaceutically active protein with recombinant DNA method.Compare with polymer-modified product, product of the present invention obviously is easy to identify.Because the employing recombinant method for production can obtain a plurality of multiple homogeneous products with clear and definite characterization of molecules.The URP sequence also can be beneficial to product purification.For example, the URP sequence can comprise the subsequence that can be caught by affinity chromatography.An example is the sequence that is rich in histidine, and its can be cured resin of metal such as nickel is caught.Can design the URP sequence, make it contain the aminoacid of too much electronegative or positive charge.So the net charge of their energy appreciable impact products, this helps by ion exchange chromatography or this product of preparation electrophoresis purification.

Theme MURP contains multiple module, includes but not limited to binding modules, effect module, multimerization module, C-terminal module and N-terminal module.The example MURP that Fig. 1 explanation has a plurality of modules.Yet MURP also can have relatively simply structure as shown in Figure 2.MURP also can comprise the fragmentation site.They can be responsive sequence of protease or chemical-sensitive sequence, and these sequences can preferentially be cut when MURP arrives its target spot.

Binding modules (BM):

MURP of the present invention can comprise one or more binding modules.Binding modules (BM) refers to the peptide or the protein sequence of energy specificity and one or more targeted integration, and described target spot can be one or more therapeutic target spots or attached target spot, as is used for the target spot of targeted cells, tissue or organ.BM can be the protein structure domain of linear peptides or cyclic peptide, cysteine constraint peptide, microprotein, scaffolding protein (as fibronectin, anchorin, crystal, Streptavidin, antibody fragment, domain antibodies), peptide hormone, somatomedin, cytokine or any type (people or inhuman, natural or non-natural), they can be based on natural support or not based on natural support, or based on its combination, the perhaps fragment of above-mentioned any material.Randomly, these BM can be by adding, removing or replace one or more aminoacid and carry out engineered to strengthen it in conjunction with attribute, stability or other character.Binding modules can be showed by design or heredity bag available from native protein, comprised that phage display, cell display, ribosomal display or other methods of exhibiting obtain.Binding modules can be incorporated into the same copy of same target spot, cause affinity or with the different copies of same target spot in conjunction with (if these copies in a way by (as) cell membrane contact or connect, can cause affinity), or they can with two uncorrelated targeted integration (if these target spots in a way by (as) symphysis connects, and causes affinity).By screening or analyze peptide or proteic random library can be identified binding modules.

In case the binding modules that especially needs is after mixing MURP, MURP produces those binding modules of required T epi-position scoring.Proteic T epi-position scoring is the logarithm value that this albumen and the most common a plurality of people MHC allele combine Kd (dissociation constant, affinity, dissociation rate), as Sturniolo, and T. etc. (1999) Nature Biotechnology 17:555) described.These scoring scopes cover at least 15 logarithm value, from about 10,9,8,7,6,5,4,3,2,1,0 ,-1 ,-2 ,-3 ,-4 ,-5 (10e ¹⁰Kd) to about-5.Is the scoring that preferred MURP produces less than pact-3.5[KKW: absolute ratio? ]

The binding modules that comprises the disulfide bond that forms by paired two cysteine residues in addition that cherishes a special interest.In some embodiments, binding modules comprises the polypeptide with homocysteine content or high disulfide bond density (HDD).The binding modules of HDD family has the cysteine residues of 5-50% (5,6,7,8,9,10,12,14,16,18,20,25,30,35,40,45 or 50%) usually, and each domain contains at least two disulfide bond and optional cofactor such as calcium or other ion usually.

The existence of HDD support allows these modules little but still take the structure of relative stiffness.Rigidity is very important for obtaining high binding affinity, protease (comprising the protease that participates in antigen processing) and heat resistance, so makes these molecules have reduced immunogenicity or do not have immunogenicity.The a large amount of hydrophobic side chains that need not most inside modules interact, and the disulfide bond framework just can fold this module.Small size also helps fast tissue infiltration and other optional route of delivery, as in oral, nasal cavity, the intestinal, pulmonary, blood brain barrier etc.In addition, small size also helps to reduce immunogenicity.The domain that number by increasing disulfide bond or utilization contain less aminoacid and same number disulfide bond can obtain higher disulfide bond density.Also need to reduce the fixedly number of residue of non-cysteine, so that make more a high proportion of aminoacid be used for targeted integration.

The binding modules that contains cysteine can take various disulfide bond to become key pattern (DBP).For example, two disulfide bond modules can have three kinds of disulfide bond and become key pattern (DBP), and three disulfide bond modules can have 15 kinds of different DBP, and four disulfide bond modules can have 105 kinds of different DBP of as many as.All 2SSDBP, most of 3SSDBP and fewer than half 4SS DBP have natural example.In one aspect, can calculate the sum that disulfide bond becomes the key pattern according to formula: mistake! Can't from the edit field code, create object, wherein the predicted number of the disulfide bond of n=cysteine residues formation, wherein mistake! Can't create the result of object representative (2i-1) from the edit field code, wherein i is the positive integer of 1-n.

Therefore, in one embodiment, the module of using among the MURP is to contain the support natural or cysteine (C) that non-natural produces, and this fingernail has binding specificity to target molecule, wherein according to being selected from the formula mistake! Can't create one group of pattern of rows and columns of object representative from the edit field code, the support that contains non-natural cysteine (C) comprises cysteine in the support, and wherein n equals the predicted number that cysteine residues forms disulfide bond, and mistake! Can't create object from the edit field code and represent product (2i-1), wherein i is the positive integer of 1-n.In one aspect, the module that contains cysteine that produces of natural or non-natural comprise have by in the polypeptide in pairs cysteine according to being selected from C ^1-2,3-4, C ^1-3,2-4Or C ^1-4,2-3The polypeptide of two disulfide bond forming of pattern, wherein two numerals that connected by hyphen are from which two cysteine pairing formation disulfide bond of polypeptide N-terminal.On the other hand, the module that contains cysteine that produces of natural or non-natural comprises and has by cysteine in the paired support according to being selected from C ^1-2,3-4,5-6, C ^1-2,3-5,4-6, C ^1-2,3-6,4-5, C ^1-3,2-4,5-6, C ^1-3,2-5,4-6, C ^1-3,2-6,4-5, C ^1-4,2-3,5-6, C ^1-4,2-6,3-5, C ^1-5,2-3,4-6, C ^1-5,2-4,3-6, C ^1-5,2-6,3-4, C ^1-6,2-3,4-5Or C ^1-6,2-5,3-4The polypeptide of three disulfide bond forming of pattern, wherein two numerals that connected by hyphen are from which two cysteine pairing formation disulfide bond of polypeptide N-terminal.On the other hand, natural or non-natural produces contains the cysteine module and contains and have by the polypeptide of at least four disulfide bond forming according to the pattern of rows and columns that is selected from above-mentioned formula definition of cysteine in pairs in the polypeptide.On the other hand, the module that contains cysteine that produces of natural or non-natural contains and has by at least five, six or the polypeptide of more a plurality of disulfide bond forming according to the pattern of rows and columns that is selected from above-mentioned formula definition of cysteine in pairs in the polypeptide.Await the reply altogether and anyly in the application [series number 11/528,927 and 11/528,950, include this paper in full for referencial use] contain cysteine protein or support is candidate's binding modules.

Have 4,5,6,7,8,9,10,11 and 12 between the cysteine of the optional comfortable disulfide-bonded of binding modules at random or the cysteine confinement ring peptide library of part random amino acid (as with the structure pattern), available in some cases several methods makes up extra random amino acid at cystine to the outside.Can utilize the whole bag of tricks, comprise that phage display, ribosomal display, yeast are showed and other method known in the art identifies that target spot interested is had specific library member.Can utilize this type of cyclic peptide as the binding modules among the MURP.One preferred embodiment in, can utilize the further engineered cysteine restriction of the construction method that makes binding modules contain above disulfide bond peptide, to improve its binding affinity, proteolysis stability and/or specificity.Figure 25 has shown a specific construction method.This method is based in that at first N-terminal one side and C-terminal one side of selected cyclic peptide add independent cysteine and a plurality of residue at random.Can produce the library designed as Figure 25.Can utilize phage display or similar technique to identify and have the binding modules that improves characteristic.This type of makes up 1-12 the random site that the library can be included in N-terminal one side and C-terminal one side of cyclic peptide.Add newly that the distance between the cysteine residues can change in the flank cysteine residues and cyclic peptide at random between 1-12 residue.Each member of this type of library comprises four cysteine residues, and two from original cyclic peptide, and two are positioned at and newly add flank.The variation of this method preference 1-42-3DBP or DBP has been broken an existing 1-2 disulfide bond (2-3 the in=4-cysteine construction) and has been formed 1-23-4 or 1-32-4DBP.Can utilize the clone-specific primer to carry out this construction method, the feasible not interregional fixed sequence program (as shown in figure 25) that stays in the library, the primer of the fixed sequence program of the peptide both sides that perhaps utilization (also therefore staying) is at first selected carries out, therefore these same primers can be used for any clone who at first selects, as shown in figure 26.Method as shown in figure 26 can be applicable to that target spot interested is had specific cyclic peptide set.Proved that these two kinds of construction methods all can play a role in the affine maturation of the anti-VEGF of structure.Repeat this method contains six or more a plurality of cysteine residues with generation binding modules.

Figure 27 has shown that another kind is constructed into a disulfide bond in the 2-disulfide bond sequence.This method comprises the 1-disulfide bond peptide storehouse and the dimerization of itself of original selection, so that preselected peptide storehouse stops at N-terminal and C-terminal position.This method is beneficial to the structure of the 2-disulfide bond sequence that makes up two different epi-positions of target spot.

Another building method comprises (part) randomized sequence that adds 6-15 the residue that comprises two cysteine, these two cysteine intermediate phase also can be chosen the additional random position wantonly every 4,5,6,7,8,9 or 10 aminoacid outside the cysteine that connects.Selecting N-terminal one side of peptide or C-terminal one side to add this 2-cysteine random sequence in advance.This method preference 1-23-4DBP is though also can form other DBP.Repeat this method contains six or more most cystine residues with generation binding modules.

Can make up binding modules according to the native protein support.Can identify this type of support by database retrieval.The phage display elutriation can be carried out in library based on natural support, screens then to differentiate the sequence of specificity in conjunction with target spot interested.

The natural support range of choice that is used to make up binding modules is very wide.The selection of particular stent depends on target.The non-limitative example of natural support comprises snake venom sample albumen, as the ectodomain of venom toxin and human cell surface receptor.The non-limitative example of venom toxin is laticotoxin B, γ-cardiotoxin, the anticholinergic neurotoxin, the muscarine toxin, semi-ring tang sea snake neurotoxin A, neurotoxin I, cardiotoxin V4II (toxin III), cardiotoxin V, α-cobratoxin, long neurotoxin venom 1, the FS2 toxin, the Bungarus fasciatus toxin, the Malaysia bungarotoxin, cardiotoxin CTXI, cardiotoxin CTXIIB, cardiotoxin II, cardiotoxin III, cardiotoxin IV, cobratoxin 2, alpha-toxin, neurotoxin II (cobratoxin B), toxin B (long neurotoxin venom), the many toxin of bank (Candotoxin), bucainide.The non-limitative example of (people) cell surface receptor ectodomain comprises CD59, II type activin acceptor, bmp receptor Ia external structure territory, TGF-β II receptor ectodomain.Other natural support includes but not limited to A-domain, EGF, Ca-EGF, TNF-R, Notch, DSL, Trefoil, PD, TSP1, TSP2, TSP3, Anato, integrin B, Elityran, defensin 1, defensin 2, plant cyclase protein, SHKT, removes integrin, myotoxin, γ-thioneine, conotoxin, mu-conotoxin, ω-spider venom toxin, δ-Atraco toxin and in co-pending application series number 11/528 altogether, 927 and 11/528,950 disclosed families, full text is included the list of references of this paper in.

Several different methods how to identify binding molecule from big variant library had been described.A kind of method is chemosynthesis.Synthetic library member on pearl, so each pearl carries different peptide sequences.Utilize the binding partner of labelling to identify the pearl that carries required ligands specific.Other method is to produce the peptide sublibrary, with repetitive process identify the specificity binding sequence (Pinilla, C. etc. (1992) BioTechniques, 13:901-905).More usually at the methods of exhibiting in the surface expression variant library of phage, albumen or cell.The common ground of these methods is that the DNA of each variant in the encoded libraries or RNA physical connection are in part.Can detect or obtain part interested like this and check order to determine its peptide sequence by DNA or RNA to connection.Those skilled in the art can utilize methods of exhibiting enrichment from big random variants library to have the library member of required binding characteristic.Usually, can from enriched library, identify variant by the single separator that has desirable characteristics in the screening enriched library with required binding characteristic.The example of methods of exhibiting be merge with the 1ac repressor (Cull, M. etc. (1992) Proc.Natl.Acad Sci.USA, 89:1865-1869), cell surface display (Wittrup, K.D. (2001) Curr OpinBiotechnol, 12:395-9).The method that cherishes a special interest is peptide at random or the albumen that is connected in phage particle.M13 phage that commonly used is (Smith, G.P. etc. (1997) Chem Rev, 97:391-410) and the T7 phage (Danner, S. etc. (2001) Proc Natl AcadSci USA, 98:12954-9).Can utilize several different methods displayed polypeptide or albumen on the M13 phage.Under many situations, the N-terminal of library sequence and M13 phage display peptide pIII merges.Phage is carried this albumen of 3-5 copy usually, so the phage in this library is carried the library member of 3-5 copy as a rule.This method is that multivalence is showed.Another kind method is that the library is showed by the phasmid of phasmid coding.By infect with helper phage the cell carry phasmid can form phage particle (Lowman, H.B. etc. (1991) Biochemistry, 30:10832-10838).This process causes the unit price displaying usually.In some cases, preferred unit price is showed to obtain the zygote of high-affinity.The next preferred multivalence displaying of other situations (O ' Connell, D. etc. (2002) J Mol Biol, 321:49-56).

The several methods that has the sequence of desirable characteristics with the phage display enrichment had been described.Can be by being incorporated into immune pipe, microwell plate, the fixing target spot interested of magnetic bead or other surface.Then, phage library contacts with fixing target spot, and the phage that lacks binding partner is by flush away, and eluting carries the phage of target spot ligands specific under multiple condition.Eluting can carry out under low pH, high pH, carbamide or other are tending towards interrupting the condition of protein-protein contact.Also can add the phage of Bacillus coli cells elution of bound, but the escherichia coli host that the wash-out bacteriophage direct infection is added.Interesting testing program is to utilize the degradable phage binding partner or the fixing protease eluting of target spot.Protease also can be used as the instrument of rich protein enzyme resistance phage binding partner.For example, before elutriation target spot interested, phage binding partner library and one or more (people or mice) protease are hatched.This process from the library, degrade and removed protease unstability part (Kristensen, P. etc. (1998) Fold Des, 3:321-8).Also can be by combine the phage display storehouse of enrichment part with complex biological sample.Example be solidify cell membrane component (Tur, M.K. etc. (2003) Int JMol Med, 11:523-7) or whole cell (Rasmussen, U.B. etc. (2002) Cancer Gene Ther, 9:606-12; Kelly, K.A. etc. (2003) Neoplasia carries out elutriation on 5:437-44).In some cases, optimize the elutriation condition with the efficient that improves enrichment of cell specific conjugate from the phage display storehouse (Watters, J.M. etc. (1997) Immunotechnology, 3:21-9).The phage elutriation also can be carried out on live body patient or animal.This method for the part that evaluation is incorporated into the blood vessel target spot interested especially (Arap, W. etc. (2002) Nat Med, 8:121-7).

Those skilled in the art can utilize several cloning process to produce the cDNA library in encoded peptide library.Can use the synthetic oligonucleotide that comprises one or more random sites of random mixture of nucleotide.The number and the degree of randomization of this process may command random site.In addition, can obtain at random or half random dna sequence by the DNA that partly digests biological sample.Can use random oligonucleotide to make up in advance but library with randomized plasmid or phage in the position.Can be by (248:97-105) described method is carried out the PCR fusion for de Kruif, J. etc. (1995) J Mol Biol.Other method connects (Felici, F. etc. (1991) JMol Biol, 222:301-10 based on DNA; Kay, B.K. etc. (1993) Gene, 128:59-65).Other common method is Kang Keer (Kunkel) mutation, wherein is the mutation chain of template synthetic plasmid or phasmid with the strand cyclic DNA.Referring to: Sidhu, S.S. etc. (2000) Methods Enzymol, 328:333-63; Kunkel, T.A. etc. (1987) Methods Enzymol, 154:367-82.

Kang Keer mutation is used and to be contained the template of mixing the uracil base at random that can obtain from coli strain such as CJ236.Contain the uracil template strand and preferably after conversion enters escherichia coli, degrade, and external synthetic mutation chain is retained.The most transformants of result carry the phasmid or the phage of mutation.Increasing the multifarious valuable method in library is to merge a plurality of sublibraries.Can generate these sublibraries by above-mentioned any means, they can be based on identical or different support.

Recently described the process useful that produces big small peptide phage library (Scholle, M.D. etc. (2005) Comb Chem High Throughput Screen, 8:545-51).This method is relevant with the Kang Keer method but need not to generate and comprise the single-stranded template of uracil base at random.In fact, this method is from carrying one or more template phagies near the sudden change of mutation zone, and described sudden change makes phage not have appeal.This method uses some position to carry the mutagenic oligonucleotide of random cipher and the sudden change of energy calibration template pnagus medius inactivation.The result is, only the mutator phage granule has infectivity after conversion, and only a few parent's phage is contained in this type of library.Can also further modify this method by several approach.For example, can utilize a plurality of mutagenic oligonucleotides a plurality of non-adjoins region of mutator phage simultaneously.We further utilize this method, are applied to〉25,30,35,40,45,50,55 and 60 amino acid whose whole microproteins, but not＜10,15 or 20 amino acid whose small peptides, this is another challenge.Therefore this method estimates that 10 conversions can obtain the single library that multiformity is 10e12 once transforming the library of back generation more than 10e10 transformant (10e11 at most).

Another modification of super power (Scholle) method is the design mutagenic oligonucleotide, makes the succinum termination codon in the template be converted into Haematitum termination codon, then is that Haematitum becomes succinum in next mutation circulation.In this case, the template phage must be incubated at different escherichia coli with mutation library member and suppress in the daughter bacteria strain, and Haematitum suppresses son and succinum suppresses the daughter bacteria strain alternately.Can change by termination codon and two kinds of successive phage mutation that suppress to hocket between the daughter bacteria strain like this at two types.

The modification of another kind of super power method relates to uses single stranded phage dna profiling and big primer.Big primer is the long ssDNA that produces the library insert of the phage library of selecting from the previous round elutriation.Purpose is to catch various libraries insert from previous storehouse, in one or more zones it is carried out mutation and can it be gone in the new library by the method for mutation with other zone.Can utilize the same template that contains gene of interest termination codon that big primer is carried out several circulation processing of repetition.Big primer is ssDNA (optional by the PCR generation), comprise 1) with 5 ' and 3 ' overlay region of complementary at least 15 bases of ssDNA template, 2) copy is from the zone, one or more original selection library (1 of (the optional PCR that passes through) original clone bank of selecting, 2,3,4 or more) and 3) zone, library of the new mutation selected in the next round elutriation.Choose wantonly and prepare big primer by the following method: the 1) oligonucleotide in the synthetic new synthetic library of one or more codings zone, 2) utilize overlapping PCR that itself and dna fragmentation are merged (obtaining by PCR alternatively) alternatively, described dna fragmentation comprises other zone, library of original optimization.Utilize (Run-off) out of control or the strand PCR that merge (overlapping) PCR product to produce the big primer of strand in the new library that comprises all original other zones of optimizing the zone and will in next round elutriation experiment, optimizing.Estimate that this method can utilize repeatedly fast the library to create circulation and carry out the affine maturation of albumen, each takes turns the multiformity that generates 10e11-10e12, carries out elutriation then.

Can use several methods calling sequence multiformity in (selection or original in advance) microprotein library, or mutation single microbial albumen clone, purpose is to strengthen its combination or other characteristic, as manufacturing, stability or immunogenicity.In principle, all methods that can be used to produce the library also are used in microprotein enrichment (the original selection) library and introduce multiformity.Specifically, can synthesize variant with required combination or other characteristic and according to these sequential design incomplete randomization oligonucleotide.This process allows randomized position of control and degree.Can utilize several computerized algorithms (Jonsson, J. etc. (1993) Nucleic Res, 21:733-9; Amin, N. etc. (2004), Protein EngDes Sel 17:787-93) is inferred the effectiveness of single sudden change in the protein by a plurality of variant sequence datas.Cherish a special interest be used for the enrichment storehouse again the method for mutation be that (370:389-391), this will produce the recon of single sequence in enriched library for Stemmer, W.P.C. (1994) Nature for DNA reorganization.Utilize the PCR condition of multiple improvement to reorganize, and the template of can partly degrading is to strengthen reorganization.Another possibility is to utilize to recombinate based on the precalculated position that is cloned in of Restriction Enzyme.Cherish a special interest be utilize can be outside the sequence recognition site IIS type Restriction Enzyme of cutting DNA method (Collins, J. etc. (2001) JBiotechnol, 74:317-38).Can use the Restriction Enzyme that can produce non-palindrome jag plasmid or other DNA at a plurality of sites cutting coding variant mixture, and by connect assemble again complete plasmid (Berger, S.L. etc. (1993) AnalBiochem, 214:571-9).Another introduces multifarious method is PCR-mutation, and the DNA sequence to the encoded libraries member under mutagenic condition is carried out PCR.Described the PCR condition that causes high relatively mutation frequency (Leung, D. etc. (1989) Technique, 1:11-15).In addition, can use the polymerase that fidelity reduces (Vanhercke, T. etc. (2005) Anal Biochem, 339:9-14).The method that cherishes a special interest is based on method (Irving, R.A. etc. (1996) Immunotechnology, the 2:127-43 of muton strain; Coia, G. etc. (1997) Gene, 201:203-9).These bacterial strains carry one or more DNA-repair gene sudden changes.Plasmid in these bacterial strains or phage or other DNA accumulate sudden change in the process of normal replication.But single clone in the strain of propagation of mutated daughter bacteria or enrichment colony are to introduce genetic diversity.Above-mentioned many methods can be used for repetitive process.Can use the mutation of many wheels and screening or elutriation to the part of whole gene or gene, or in each subsequent passes to proteic different piece carry out mutation (Yang, W.P. etc. (1995) J Mol Biol, 254:392-403).

Further handle the library to reduce pseudomorphism.The known pseudomorphism of phage elutriation comprises 1) based on hydrophobic non-specific binding, 2) combine with the multivalence of target spot, reason is an a) pentavalent pIII phage albumen, or b) disulfide bond that forms between different microorganisms albumen, produce polymer, or c) high density coatings of target spot on the solid support, and the 3) targeted integration that relies on of environment (context), the environment (context) of environment of its point of impact on target (context) or microprotein in conjunction with or suppress active most important.Take different treatment steps to reduce to minimum with amplitude with these problems.For example, this type of processing can be applicable to whole library, but some useful processing that remove bad clone only can be applied to soluble protein storehouse or single soluble protein.

Free sulfhydryl groups may be contained in the library that contains the cysteine support, and sulfydryl is by making orthogenic evolution become complicated with other protein-crosslinking.A kind of method is to remove the worst clone in the library by flowing through the free sulfhydryl groups post, therefore can remove all clones with one or more free sulfhydryl groups.The clone that free SH group arranged also can with biotin-SH reagent reacting so that use the Streptavidin post effectively to remove the clone of responding property SH group.Other method is not remove free sulfhydryl groups, but can utilize sulfydryl reactive compounds such as iodoacetic acid to add medicated cap and make its inactivation.What cherish a special interest is high capacity or the hydrophilic sulfhydryl reagent that reduces non-specific binding or modify variant.

Environment (context) dependency example is all constant serieses, comprises that pIII albumen, joint, peptide tag, biotin-Streptavidin, Fc and other participate in interactional fusion rotein.Avoid the typical method of environmental factor dependence to comprise that as far as possible changing environment is to avoid making up (buildup) continually.Can comprise change between the different display systems (be M13 to T7, or M13 is to yeast), change used label and joint, change fixedly (solid) holder of usefulness (as fixing chemistry) and change target protein (different suppliers and different fusions) itself.

Also can use the library processing selecting to have the albumen of preferred characteristics.A kind of selection is exactly to utilize the Protease Treatment library to remove unstable variant from the library.Employed protease is generally the protease that runs among those the application.In order to carry out pulmonary delivery, can use (for example) to irritate the lung protease that lung obtains.Similarly, can obtain mixture from the protease of serum, saliva, stomach, intestinal, skin and nasal cavity etc.[adnexa E] seen in the protease tabulation on a large scale.Exceptionally, phage itself has resistance to most of protease and harsh treatment conditions.

For example, may at first remove least stable structure by being exposed to the reproducibility reagent (being DTT or β mercaptoethanol) that concentration increases progressively, select the library of rock-steady structure, promptly those have the library of the strongest disulfide bond.Usually use reproducibility reagent (being DTT, BME and other reagent) concentration as 2.5mM, to 5mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or even 100mM, this depends on required stability.

Also may then reoxidize the albumen library gradually to rebuild disulfide bond by reduce whole display libraries with high-level Reducing agent, remove the clone who contains free SH group then, selecting can external effectively folding clone.Can repeat this process of one or many to eliminate the low clone of external folding efficiency.

A kind of method is to use genetic method to select protein expression level, folding and dissolubility, as described in (Genetic selection for protein solubility enabled by the folding qualitycontrol feature of the twin-arginin translocation pathway) the Protein Science (online) that " utilizes the heredity of the protein solubility that the folding Quality Control characteristic of double arginine shift path carries out to select " such as A.C.Fisher (2006).Carry out display libraries elutriation (optional) afterwards, can avoid screening thousands of clones' targeted integration, expression and folding at protein level.Another scheme is that whole selected insert storehouse is cloned in the beta lactamase fusion vector, and when bed board on beta-lactam, the author proves that it is to expressing the selectivity of soluble protein good, complete disulfide bonding.

In M13 phage display albumen library with once or after the circulation target spot elutriation for several times, there are several approach to proceed, comprise that (1) utilizes phage E LISA to screen individual phage clone, this can measure and be incorporated into the fixedly number of the phage particle of target spot (utilizing anti--M13 antibody); (2) transfer to the T7 phage display library by M13.Second method is particularly useful aspect the false-positive incidence rate of valence state in minimizing.Any library form is tending towards preference can form the clone that high affinity contacts with target spot.Although very slow, this is a very important reasons of screening soluble protein.The multivalence that the T7 phage display forms is quite different with formation during M13 shows, and the circulation between T7 and M13 can be used as the excellent method of minimizing based on the false positive incidence rate of valence state.

It is the method that another kind is used in High Density Cultivation bacterial clump (10e2-10e5) on the big agar plate that filter membrane promotes (filter lift).A spot of some albumen is entered culture medium and finally is incorporated into (NC Nitroncellulose or nylon) on the filter membrane by secretion.In skim milk, 1% casein hydrolysate or 1%BSA solution, seal filter membrane, cultivate with the target protein of fluorescent dye or indicator enzyme (direct or indirect) labelling then by antibody or biotin-Streptavidin.By filter membrane being tiled in the position that bacterium colony is determined at the dull and stereotyped back side, selecting all positive bacterium colonies, and be used for other evaluation.The advantage that filter membrane promotes is and can reads signal and it has been designed to affine selectivity by the different periods after washing.High-affinity clone's signal " decay " is slow, and low-affinity clone's signal is then decayed soon.This type of affine evaluation usually needs 3 experiments and based on the experiment in hole, and with the comparability that provides between better clone-clone is provided based on the experiment in hole.It is the minimized process useful of difference that bacterium colony size and position are brought that bacterium colony is divided into grid array.

The N-terminal module:

Theme MURP can comprise N-terminal module (NM), is promoting MURP particularly useful in producing.When product was expressed in Bacillus coli cells matter, NM can be single methionine residues.The form of typical product is the URP that merges with human cytokines, and it is a formylmethionine at this N-terminal of antibacterial kytoplasm invading the exterior dyne.Formylmethionine can be permanent or interim (if being removed by biological or chemical processing).

NM carries out engineered peptide sequence for the proteolysis process, can be used for removing label or removes fusion rotein.Through engineered, the N-terminal module can be by comprising the purification that affinity tag such as Flag-, Myc-, HA-or His-label are beneficial to MURP.The N-terminal module also can comprise the affinity tag that is used to detect MURP.Can carry out NM engineered or select with high level expression MURP.Also can be engineered or select to strengthen the protease resistant of gained MURP with it.Can produce the MURP of N-terminal module with the expression of being beneficial to and/or purification.Utilize protease the N-terminal module can be cut in process of production, so that end product does not comprise the N-terminal module.

Select to increase reorganization output by aminoacid and the codon of optimizing the N-terminal module.The N-terminal module also can comprise can be by the processing site of specific proteases such as Xa factor, thrombin or enterokinase, the cutting of Fructus Lycopersici esculenti etch virus poison (TEV) protease.But the Design and Machining site is so that cut by chemical hydrolysis.An example is the aminoacid sequence asp-pro that can be cut under acid condition.Also can design the N-terminal module that is beneficial to the MURP purification.For example, can design the N-terminal module and comprise a plurality of his residues, so that catch product by the fixing metal chromatograph.The N-terminal module can comprise the energy specificity by the peptide sequence of antibody capture or identification.Example is FLAG, HA, c-myc.

The C-terminal module:

MURP can be included in and promote useful especially C-terminal module in the MURP production.For example, thus the C-terminal module can comprise carries out proteolysis processing and removes the cleavage site that fusion sequence improves protein expression or promotes purification.Specifically, also can comprise can be by the processing site of specific proteases such as Xa factor, thrombin, TEV protease or enterokinase cutting for the C-terminal module.The processing site that design can be cut by chemical hydrolysis.Example is the aminoacid sequence asp-pro that can cut under acid condition.The C-terminal module can be the affinity tag that is beneficial to the MURP purification.For example, can design the C-terminal module so that it comprises a plurality of his residues, so that catch product by the fixing metal chromatograph.The C-terminal module can comprise the energy specificity by the peptide sequence of antibody capture or identification.The non-limitative example of label comprises FLAG-, HA-, c-myc or His-label.Also can be engineered or select the C-terminal module to strengthen the protease resistant of gained MURP.

Required is, this proteic N-terminal can be linked to each other with himself C-terminal.For example, by creating the natural connection of aminoacid sample (peptide bond) or using exogenous connector that these two modules are coupled together.What cherish a special interest is cyclic peptide, and it is the small protein family of the natural generation of a class.Estimate to adopt the version of similar cyclic peptide that the more stability of resisting exogenous protease can be provided.Connecting in than this quasi-molecule under the low protein concns to work better.

The effect module:

MURP can comprise one or more effect modules (EM) or not have the effect module at all.The effect module does not provide targeting usually, but provides therapeutic effect required activity, as cell killing.EM can be pharmaceutical active micromolecule (being drug toxicity), peptide or albumen.Non-limitative example is complete cytokine, abzyme, somatomedin, hormone, receptor, receptor stimulating agent or antagonist, or its fragment or domain.The effect module also can comprise carries the peptide sequence that synthetic or natural chemistry connects small-molecule drug.Alternatively, these effector molecules can be connected with the effect module by chemical joint, can cut or not cut these joints under selected condition, thereby discharge the toxicity activity.EM also can comprise radiosiotope and chelate thereof, and the different labels that are used for PET and MRI detection.But the effect module is pair cell or organize poisonous also.Cherish a special interest be comprise the poisonous effect module and with the MURP of diseased tissue or the bonded binding modules of disease cell type specificity.This type of MURP can accumulate in diseased tissue or disease cell by specificity, and preferentially brings into play its toxic action in the disease cell or tissue.Classify the example of effect module down as.

Enzyme-effect module can be enzyme.What cherish a special interest is the crucial metabolite of degraded cell growth, as the enzyme of sugar or aminoacid or fat or cofactor.Other example with effect module of enzymatic activity is RNA enzyme, DNA enzyme and phosphatase, asparaginase, histidase, arginase, beta lactamase.Effect module with enzymatic activity can have toxicity when being delivered to tissue or cell.That cherish a special interest is the MURP that poisonous effect module and specificity are combined in conjunction with the binding modules of diseased tissue.Can also be potential effect module with the enzyme that nonactive prodrug is converted into active medicine at tumor sites.

Medicine-theme MURP can comprise medicine action effect thing.Required is, can be designed for the sequence that the organ selectivity of drug molecule is sent.An example is seen Fig. 8.The URP sequence can merge with the albumen of preferred combination in diseased tissue.Same URP sequence can comprise modified can with the bonded one or more amino acid residues of drug molecule.This type of conjugate is incorporated into diseased tissue with high degree of specificity, and the drug molecule that is connected can produce local action, thereby the whole body contact of cell drug is reduced to minimum.Can design the release that MURP is beneficial to drug molecule at required action site by the responsive site of protease that neutral protease cuts by introducing at the target spot place.The significant advantage that utilizes URP sequential design medicine to send construction is the bad interaction that can avoid between drug molecule and the construction targeting domain.Can have significant hydrophobicity with the link coupled many drug molecules of targeting domain, make that the gained conjugate is tending towards assembling.Can improve the dissolubility that gained is sent construction by hydrophilic URP sequence is joined this type of construction, thereby reduce aggregation tendency.And, can increase the number of the drug molecule that merges with the targeting domain by adding long URP sequence.In addition, the distance of using the URP sequence can optimize between the drug coupling site is beneficial to finish coupling.The medicine tabulation that is fit to includes but not limited to chemotherapeutic, as: thiotepa and cyclophosphamide (cyclophosphamide ^TM); Alkyl sulfonic acid is as busulfan, an improsulfan and piposulfan; Ethylene imine is as benzodepa, carboquone, meturedepa and uredepa; Aziridine and first melamine comprise altretamine, tretamine, triethylenephosphoramide, triethylene D2EHDTPA amine and trimethylolmelamine; Chlormethine is as chlorambucil, chlornaphazine, chlorine phosphamide, estramustine, ifosfamide, chlormethine, mustron, melphalan, novembichin, NSC-104469, prednimustine, trofosfamide, uracil mustard; Nitroso ureas is as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, Ranimustine; Antibiotic is as aclarubicin, D actinomycin D, antramycin, azaserine, bleomycin, D actinomycin D c, calicheamicin, OK a karaoke club is than star (carabicin), carminomycin, cardinophyllin, chromomycin, D actinomycin D d, daunomycin, detorubicin, 6-diazonium-5-oxo-L-nor-leucine, doxorubicin, epirubicin, esorubicin, the jaundice element, the Marcelo mycin, mitomycin, mycophenolic acid, nogalamycin, Olivomycin, peplomycin, Bo Feiluo mycin (potfiromycin), puromycin, triferricdoxorubicin, rodorubicin, rufocromomycin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; Antimetabolite is as methotrexate and 5-fluorouracil (5-FU); Folacin is as 9,10-dimethylpteroylglutamic acid, methotrexate, Pteropterin, trimetrexate; Purine analogue is as fludarabine, Ismipur, ITG, thioguanine; Pyrimidine analogue is as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, di-deoxyuridine, doxifluridine, enocitabine, floxuridine; Androgen such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, Testolactone; Antiadrenergic drug is as aminoglutethimide, mitotane, trilostane; The folic acid fill-in is as the leaf alkanoic acid; Aceglatone; The aldophosphamide glucosides; Amino-laevulic acid; Amsacrine; Beta cloth suffering (bestrabucil); Bisantrene; Yi Da Qu Sha; Defosfamide (defofamine); Demecolcine; Diaziquone; Many Ka-7038s (duocarmycin), maylasine (maytansin), Er Tating (auristatin), Ai Fomixin (elfomithine); According to sharp vinegar amine; Etoglucid; Ganite (Fujisawa).; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; C-283; Pentostatin; Phenamet; Pirarubicin; Podophyllinic acid; 2-ethyl hydrazides; Procarbazine; PSK.R ^TMRazoxane; Sizofiran; Spirogermanium; Tenuazonic acid; Triaziquone; 2,2 ', 2 ＂-trichlorine triethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Add cytosine; Cytosine arabinoside (" Ara-C "); Cyclophosphamide; Thiotepa; Taxane is as paclitaxel (paclitaxel ^TM, the Bristol Myers Squibb tumor department of the Chinese Academy of Sciences, Princeton, New Jersey) (Bristol-Myers Squibb Oncology, Princeton, N.J.)) and docetaxel (taxotere ^TM, Rhone-Poulenc Rorer, Anthony, France is economized) (Antony, France)); Chlorambucil; Gemcitabine; The 6-thioguanine; Purinethol; Methotrexate; Platinum analogs is as cisplatin and carboplatin; Vinblastine; Platinum; Etoposide (VP-16); Ifosfamide; Ametycin; Mitoxantrone; Vincristine; Vinorelbine; Nvelbine; Dihydroxyanthraquinone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; Camptothecine-11 (CPT-11); Topology isomerase inhibitors RFS2000; Er Fujiajiniaoansuan (DMFO); Tretinoin; Dust draws mycin (esperamicins); Capecitabine; Pharmaceutically acceptable salt, acid or derivant with above-mentioned substance.The suitable chemotherapy cell modulator that also comprises, they be used to reconcile or inhibitory hormone to the hormone antagonist reagent of function of tumor, as anti-estrogen, comprise as tamoxifen, raloxifene, aromatase and suppress 4 (5)-imidazoles, 4-trans-Hydroxytamoxifen, trioxifene, raloxifene hydrochloride, LY117018, onapristone and toremifene (Fareston); And androgen antagonist, as flutamide, nilutamide, bicalutamide, leuprorelin, goserelin, doxorubicin, daunomycin, many Ka-7038s (duocarmycin), vincristine and vinblastine.

Other can be used as the effect module medicine comprise be used for the treatment of catch, heart disease, infectious disease, respiratory disorder, autoimmune disease, N﹠M are not normal, the medicine of metabolism disorder and cancer.

Other medicine that can be used as the MURP effector comprises: unsting medicine and antibiotic medicine, as histamine and histamine antagonist, Kallidin I and brad ykinin antagonists, serotonine (5-hydroxy tryptamine), the lipid that product produced of biotransformation selective hydrolysis membrane phospholipid, eicosanoid, prostaglandin, thromboxane, leukotriene, aspirin, non-steroid anti-inflammatory agent, the acetaminophen medicine, suppress prostaglandin and the synthetic medicine of thromboxane, the selective depressant of inductivity cyclo-oxygenase, the selective depressant of inductivity cyclo-oxygenase 2, autacoid, the paracrine hormone, somatostatin, gastrin, the interactional cytokine of mediation in body fluid and cellullar immunologic response, the deutero-autacoid of lipid, eicosanoid, beta-adrenaline excitant, ipratropium, glucocorticoids, methylxanthine, sodium channel blockers, opioid receptor agonist, calcium channel blocker, membrane stabilizer and leukotriene inhibitors.

Other medicine as effector comprises the medicine for the treatment of peptic ulcer, medicine, digestive tract power medicine, Bendectin, irritable bowel syndrome medication, diarrhoea medication, constipation medication, inflammatory bowel medication, the sick medication of bile and the sick medication of pancreas of treatment gastroesophageal reflux disease.

Radionuclide-design MURP is used for organizing targeted delivery and utilizing radionuclide imaging of radionuclide.Can optimize the half-life by changing URP length, so URP is the ideal material of imaging.In most imaging applications, URP that may preferred moderate-length, the half-life be 5 minutes to a few hours but not several days to several weeks.What design MURP made that it only contains single or a small amount of quantification can be by the amino of the chelating agen (as DOTA) of radiosiotope such as technetium, indium, yttrium (expansion) modification.Another kind of coupling method is by conservative cysteine side chain coupling.Can use this type of MURP that carries radionuclide treatment tumor or other diseased tissue, and be used for imaging.

Many pharmaceutically active proteins or protein structure domain can be used as the effect module among the MURP.For example following albumen and fragment thereof: cytokine, somatomedin, enzyme,-receptor, microprotein, hormone, erythropoietin (erythopoetin), adenosine takes off the imines enzyme, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, the TNF-receptor, uricase, rasburicase, Axokine, the RNA enzyme, the DNA enzyme, phosphatase, Pseudomonas exotoxin, ricin, gelonin, the general enzyme of ammonia, the La Luoni enzyme, thrombin, thrombin, VEGF, general Lip river tropine (protropin), growth hormone, alteplase, interleukin, the IIV factor, the VIII factor, the X factor, the IX factor, streptodornase, glucocerebrosidase, follitropin, glucagon, thyrotropin, Nesiritide, alteplase, parathyroid hormone, Ah add'sing carbohydrase, the La Luoni enzyme, methioninase.

Proteinase activated MURP: in order to improve the therapeutic index of effect module, the unstable sequence of protease can be inserted in the URP sequence, this URP sequence is for the preferential protease sensitivity of finding in the target tissue of serum or MURP treatment.This method is seen Fig. 9.Some designs can make up selectively activated albumen when arriving target tissue.What cherish a special interest is at the activated MURP of disease site.Activate in order to be beneficial to this type of target spot specificity, the URP sequence can be connected near the avtive spot or receptor binding site of effect module, the fusion rotein biologic activity that obtains thus is limited.What cherish a special interest is in tumor sites activation effect module.Many tumor tissues can be inserted into the sequence of these oncoprotein enzyme spcificity cuttings in the URP sequence with high relatively concentration expressing protein enzyme.For example, most prostate tumor tissues contain the prostate specific antigen (PSA) of high concentration, and this is a serine protease.By the prodrug of forming with the link coupled PSA unstability of cancer therapy drug doxorubicin peptide can be in prostata tissue selective activation [DeFeo-Jones, D. etc. (2000) Nat Med, 6:1248].It is to have cell growth inhibiting activity or Cytotoxic albumen that the disease specific that cherishes a special interest activates, as TNF α and many cytokines and interleukin.Another application is in the inflammation site or the albumen selective activation in virus or bacterial infection site.

Production method-utilize molecular biology method well known in the art can produce the MURP that comprises the URP sequence.Several cloning vehicles can be used for various expression systems, as mammalian cell, yeast and microorganism.The expressive host that cherishes a special interest is escherichia coli, saccharomyces cerevisiae, Pichia sp. and Chinese hamster ovary cell.What cherish a special interest is through optimizing the host that its codon of expansion uses.What cherish a special interest is to strengthen the host that GRS expresses through modifying.Can reach this purpose by the DNA that coding glycine specificity tRNA is provided.In addition, can engineered host to increase the carrying capacity of glycine specificity tRNA.Coding can be strengthened proteic DNA and be operatively connected to promoter sequence.The part that the promoter that coding proteic DNA of enhancing and operability connect can be used as plasmid vector, viral vector maybe can be inserted in host's the chromosome.

Under the condition that is beneficial to the enhancing protein production, cultivate host cell to produce.What cherish a special interest is the condition that can improve GRS output.

Theme MURP can adopt several forms.For example, thus MURP can comprise the URP that merges with pharmaceutically active protein obtains the slow release product.This type of product can be by local injection or implantation, for example patient skin or under skin.Because its big hydrodynamic radius, the product that contains the URP sequence slowly discharges from injection or implantation site, thus the frequency that has reduced injection or implanted.Can design the URP sequence, make it comprise zone with cell surface or tissue bond with the local holdup time of prolong drug in injection site.What cherish a special interest is that the product that will contain URP is made gathering of injection back or sedimentary soluble compounds.The pH that changes formulation products and injection site can trigger and assemble or precipitation.Another selection is can be by changing the product that redox environment causes precipitation or forms the accumulative URP of containing.Another kind method is stable in solution by adding nonactive solute, but the injection back is because the diffusion of solubilising solute causes the product of precipitation or the accumulative URP of containing.Another kind method is that design has one or more lysines or cysteine residues and can be before the injection crosslinked product that contains URP in the URP sequence.

Required is, when manufacturing, preparation and injection, MURP is monomer (referring to uncrosslinked here), but behind subcutaneous injection, albumen begin self-crosslinking or with the natural human protein-crosslinking, under skin, form polymer, active drug molecule very discharges slowly.This release can be disulfide bond reduction or disulfide bond reorganization shown in Figure 180, or by proteolysis mediation shown in Figure 19, discharges active fragment in circulation.It is highly important that these active fragments are enough big, to obtain the long half-lift, because the secretion half-life is long more, it is low more to discharge proteic dosage, can use like this than the product of low dosage inject or injection interval longer.

A kind of method of bringing these advantages is the protein-crosslinking of disulfide bond mediation.For example, can make the protein drug that contains (one or more) cyclic peptide.This cyclic peptide can participate in or not participate in the combination of target spot.Make this albumen, form cyclic peptide, i.e. the albumen of oxidised form is to simplify purification.Yet reduzate also keeps also ortho states of albumen by preparation.It is highly important that at the low concentration Reducing agent, as 0.25,0.5,1.0,2.0,4.0 or 8.0mM dithiothreitol, DTT or β mercaptoethanol or cysteine or be equal to reduce cyclic peptide in the Reducing agent, therefore can be in that other contains and reduces cyclic peptide under the condition of the albumen module of disulfide bond in the reduzate not.The preferred Reducing agent that uses the FDA approval is as cysteine or glutathion.Behind the subcutaneous injection, low-molecular-weight Reducing agent rapid diffusion or neutralized by people's albumen is exposed in the oxidation environment medicine when high molar concentration, cause the cysteine in the different protein chains crosslinked, causes the polymerization of medicine in injection site.The distance of cysteine is far away more in the cyclic peptide, and drug level is high more, and the polymeric degree of medicine is high more, because the reformation of polymerization and cyclic peptide (reformation) competition.After a period of time, the reduction of disulfide bond and oxidation cause disulfide bond reorganization, cyclic peptide is reformed and singulation, and medicine is dissolved again.Can cut the site by establishment serum albumin enzyme action comes targeting, control or increases medicine through the release of proteolysis effect from polymer.Can utilize chemical protein-protein cross-linking agent to carry out protein-crosslinking, listed as [table x].Desirable (chemical cross-linking agent) reagent for having been ratified by FDA is used for the reagent of vaccine or chemical compound and albumen coupling as those.

Except utilizing disulfide bond, also can use the effect of a variety of cross-linking agent stabilize proteins antagonism proteasome degradation.Following most reagent is sold with same name by Pierre's Si chemical company (Pierce Chemicals), and its operation instructions can obtain (www.piercenet.com) from network.Cause can be used for this application with the reagent of the same chain spacing of disulfide bond gained.Short circuit head reagent such as DFDNB are optimal.Be easy to measure the chain spacing from compound structure shown in www.piercenet.com.

A large amount of aggressiveness chemical productss play a role with following minority fundamental reaction scheme, and all the elements are all referring to the detailed description on the www.piercenet.com.Useful cross-linking agent example is imidazoles, reactive halogen, maleimide, two thiopyridines, NHS ester.The homology bi-functional cross-linking agent has two identical reactive groups and goes on foot in the chemical crosslinking step through being usually used in one.For example, BS3 (the non-water solublity DSS analog that cuts), BSOCOES (base is reversible), DMA (oneself two imido dimethyl esters, two hydrochloric acid), DMP (heptamethylene diamine dimethyl ester hydrochloric acid), DMS (hot dinitric acid dimethyl ester two hydrochloric acid), DSG (the 5 carbon analog of DSS), DSP (Luo Mante reagent (Lomant ' s reagent)), DSS (non-cut), DST (can oxidized dose of cutting), DTBP (dimethyl 3, the two third imidic acid dimethyl ester, two hydrochloric acid of 3 '-dithio), DTSSP, EGS, sulfo group-EGS, THPP, TSAT, DFDNB (1,5-two fluoro-2, the 4-dinitro benzene) particularly useful (Komblatt when crosslinked between little space length, J.A. and Lake, D.F. (1980), utilize the crosslinked cytochrome oxidase subunit of difluorodinitrobenzene (Cross-linking of cytochrome oxidase subunits with difluorodinitrobenzene), Can J.Biochem.58,219-224).

The reactive homology bi-functional cross-linking agent of sulfydryl be with the sulfydryl reaction often based on the homology bifunctional protein cross-linking agent of maleimide, when pH6.5-7.5, form stable thioether and be connected with-SH radical reaction.BM[PEO] 3 are one 8 atom polyethers spacers, can reduce that conjugate is sedimentary in the crosslinked application of sulfydryl-sulfydryl may.BM[PEO] 4 similar but have the spacer of 11 atoms.BMB is a non-cross-linking agent that cuts, and it has four carbon spacers.BMDB forms the connection that can be cut by periodate.BMH is a difunctional sulfydryl reactant cross-linker of widely used homology.BMOE has a short especially joint.DPDPB and DTME are the cross-linking agent that can cut.HVBS does not have the hydrolysis potential of maleimide.TMEA is another selection.The allos bi-functional cross-linking agent has two different reactive groups.Example is by the activatory NHS ester of EDC and amine/hydrazine, AEDP, ASBA (photoreactivity, but iodate (iodinatable)), EDC (water-soluble carbodiimide).Amine-sulfydryl reaction double functional cross-link agent is AMAS, APDP, BMPS, EMCA, EMCS, GMBS, KMUA, LC-SMCC, LC-SPDP, MBS, SBAP, SIA (short especially), SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, sulfo group-EMCS, sulfo group-GMBS, sulfo group-KMUS, sulfo group-LC-SMPT, sulfo group-LC-SPDP, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC, sulfo group-SMPB.Amino-reactive allos bi-functional cross-linking agent is ANB-NOS, MSA, NHS-ASA, SADP, SAED, SAND, SANPAH, SASD, SFAD, sulfo group-HSAB, sulfo group-NHS-LC-ASA, sulfo group-SADP, sulfo group-SANPAH, TFCS.

A different slow release form contains the medicine of His6 label, and it mixes with nickel-NTA nitrile triacetic acid-link coupled pearl (Ni-NTA pearl) and common injection, and the pearl of GMO version can be available from Kai Jie company (Qiagen).As shown in figure 20, drug slow comes off (teach off) from pearl, and bank and slow release are provided.Pearl is optionally, and the cross-linked polymeric nickel-NTA nitrile triacetic acid that can be able to be assembled into bigger polymer substitutes.

The URP sequence can comprise knownly can form polymer such as α 2D[Hill, R. etc. (1998) J Am ChemSoc, 120:1138-1145] sequence, be used to make antibody fragment dimerization [Kubetzko, S. etc. (2005) MolPharmacol, 68:1439-54].The example of useful homologous dimerization peptide is sequence SKVILFE.The example of useful allos dimerization sequence is peptide ARARAR, and it can form dimer with sequence D ADADA and correlated series.Multimerization can improve the biological function of molecule by increasing affinity, and can influence pharmacokinetic property and the tissue distribution of gained MURP.

" the multimerization module is to be beneficial to MURP to form dimer or polymeric aminoacid sequence.But multimerization module self is in conjunction with forming dimer or polymer.Perhaps, the multimerization module also can combine with other module of MURP.These modules can be leucine zipper or form the little peptide of antiparallel homologous polymerization thing such as Hydra activator derivant (SKVILF-sample), or form peptide such as the RARARA and the DADADA of highly affine anti-phase parallel allos polymer.Use one, the peptide of two or more copies can force to form protein dimer, linear polymer or branch polymer.

Can change binding affinity by type, length and the composition that changes peptide.Many application needs form the peptide of homodimer as shown in figure 21.Other application need heterodimer.In some cases, in case combination can be locked in peptide on the position by (common either side at peptide) formation disulfide bond between two protein chains.The multimerization module can be used for connecting two MURP molecules (head is to tail, head to head or end to end to tail) as shown in figure 21.For forming dimer, the multimerization module can be positioned at N or C-terminal.If two ends all have the multimerization module, then form long linear polymer.If occur two above multimerization modules in each albumen, then can form the branching polymerization network.As shown in figure 23, the design of multimerization capable of being combined and chemical coupling forms, causes that active medicine slowly discharges from bank or injection site for use in prolong half-life, stock.

Theme MURP can mix heredity or general URP.A kind of method is to express to contain the URP of long URP module (half-life is provided), and comprises permission and the peptide that combines specific target spot (i.e. linearity, ring-type, 2SS, 3SS etc.) locus specificity link coupled a plurality of (4-10 usually) lysine (or other site).The advantage of this method be the URP module be heredity and can with other any target spot specific peptide coupling.Ideal, the target spot specific peptide is direct connection with being connected of URP, therefore the residue on the URP only can with the residue reaction of target spot specific peptide, and limit coupling (exhaustive coupling) can only produce single kind, i.e. the URP that is connected with peptide on each lysine.This complex behavior is in conjunction with the similar high affinity polymer of properties, but is easy to produce.This method is referring to Figure 24.

Theme MURP also can mix URP, organizes sending of barrier so that stride.Can organize sending of barrier with enhancing leap skin, oral cavity, mouthful cheek, small intestinal, nose, blood-brain, pulmonary, sheath, peritoneum, rectum, vagina perhaps many other by engineered URP.

The major obstacle that oral protein is sent is the protease sensitivities of most albumen for digestive system.Can improve pharmaceutically active protein to resistance towards proteases and be beneficial to its picked-up with the coupling of URP sequence.Shown by adding molecular vehicle and can improve the picked-up of albumen in digestive system.The main task of these carriers is to improve permeable membrane [Sto1l, B.R. etc. (2000) J Control Release, 64:217-28].Therefore sequence can be included in and improve in the dialytic URP sequence.Known many sequences can improve permeable membrane, for example rich arginine sequence [Takenobu, T. etc. (2002) Mol Cancer Ther, 1:1043-9].Therefore can design the URP sequence that improves proteic cell or orally ingestible by the permeable membrane of two kinds of functions of combination, the proteasome degradation that reduces proteins of interest and increase fusion product.Alternatively, can be with responsive but the stable sequence of digestive tract protease is added in the URP sequence to the protease of the target tissue that is preferably placed at medicine interested.The example of this type of URP sequence is the sequence that comprises long GRS zone, and is rich in especially arginine and be beneficial to the sequence of film transhipment of basic amino acid.Can utilize URP by similar approach, to improve albumen in intranasal, lung or the picked-up of other route of delivery.

Aggressiveness product example:

DR4/DR5 agonist-DR4 and DR5 are the death receptors that is expressed in many tumor cells.Can trigger these receptors by trimerizing, cause cell death and tumor regression.Can obtain the specificity of DR4 or DR5 in conjunction with the territory by phage elutriation or other methods of exhibiting.As shown in figure 12, utilize the URP module can be with these DR4 or DR5 specificity in conjunction with the territory multimerization as joint.What cherish a special interest is to comprise three or more DR4 or DR5 or both are had the MURP of specific binding modules.As shown in figure 12, the MURP tumor antigen that can comprise overexpression in the tumor tissues has specific other binding modules.So then can make up the MURP that specificity is accumulated in tumor tissues and triggered cell death.MURP can comprise the module in conjunction with DR4 or DR5.What cherish a special interest is to contain the MURP of while in conjunction with the binding modules of DR4 and DR5.

Cancer target interleukin-22-interleukin-22 (IL2) is the cytokine that strengthens the tumor tissues immunne response.Yet the feature of general IL2 treatment is its pronounced side effects.As shown in figure 13, can make up and comprise the MURP that tumor antigen is had specific IL2 in conjunction with territory and action effect module.This type of MURP can accumulate in tumor tissues by selectivity, and causes the tumor-selective immunne response when at utmost reducing the cytokine therapy systemic side effects.But these type of several tumor antigens of MURP targeting such as EpCAM, Her2, CEA, EGFR, Thomson Fu Laide Leech antigen (Thomsen Friedenreich Antigen).That be particularly useful is the MURP that is incorporated into the tumor antigen that shows slow internalization.Can use other cytokine or tumor necrosis factor action effect module, design class is like MURP.

Tumor-selective asparaginase-use agedoite enzyme treatment acute leukemia people.Asparaginase from escherichia coli and owen bacteria (Erwinia) all can be used for treatment.Two kinds of enzymes all can cause immunogenicity and allergy.High Caspar (Oncaspar) is the asparaginase of PEGization, has the immunogenicity of reduction.Yet this albumen is difficult to make and carry out administration with the isotype mixture.Endways and/or the inner URP of adding of ring sequence allow directly a kind of agedoite enzyme variants of reorganization manufacturing, this variant be homogenizing and have a low immunogenicity.More different URP sequences and the optimum position of connection site to determine that the URP sequence is connected.Multiple other enzyme degradable is in the news and has the aminoacid of anti-tumor activity.Example is arginase, methioninase, PAL and E.C. 4.1.99.1.What cherish a special interest is the PAL of marine streptomyces (streptomycesmaritimus), and it has high activity specific and need not cofactor [Calabrese, J.C. etc. (2004) Biochemistry, 43:11403-16].Most these enzymes are antibacterial or other inhuman source and may cause immunoreation.Add one or more URP sequences and can reduce the immunogenicity of these enzymes.In addition, connect therapeutic index and the PK character that the hydrodynamic radius that increases after the URP sequence can improve these enzymes.

But design motif MURP targeting arbitrary cell albumen.Provide non-limiting tabulation below.

VEGF, VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, EGF-1, EGF-2, EGF-3, A3, cMet, ICOS, CD40L, LFA-1, c-Met, ICOS, LFA-1, IL-6, B7.1, B7.2, OX40, IL-1b .TACI, IgE, BAFF or BLys, TPO-R, CD19, CD20, CD22, CD33, CD28, IL-l-R1, TNF α, TRAIL-R1, complement receptor 1, FGFa, osteopontin, vitronectin, Ephrin A1-A5, Ephrin B1-B3, α-2-macroglobulin, CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CCL13, CCL14, CCL15, CXCL16, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, PDGF, TGFb, GMCSF, SCF, p40 (IL12/IL23), IL1b, IL1a, IL1ra, IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL23, Fas, FasL, the Flt3 part, 41BB, ACE, ACE-2, KGF, FGF-7, SCF, nerve growth factor (netrin) 1,2, IFNa, b, g, Guang winter enzyme 2,3,7,8,10, ADAMS1, S5,8,9,15, TS1, TS5; Adiponectin, ALCAM, ALK-1, APRIL, annexin V, angiogenin, amphiregulin, angiogenesis hormone-1,2,4, B7-1/CD80, B7-2/CD86, B7-H1, B7-H2, B7-H3, Bcl-2, BACE-1, BAK, BCAM, BDNF, bNGF, bECGF, BMP2,3,4,5,6,7,8; CRP, cadherin 6,8,11; Cathepsin A, B, C, D, E, L, S, V, X; CD11a/LFA-1, LFA-3, GP2b3a; the GH receptor, RSVF albumen, IL-23 (p40; p19); IL-12, CD80, CD86; CD28, CTLA-4, α 4 β 1; α 4 β 7, TNF/ lymphotoxin, IgE; CD3, CD20, IL-6; IL-6R, BLYS/BAFF, IL-2R; HER2, EGFR, CD33; CD52, digoxin, Rho (D); chickenpox, hepatitis, CMV; tetanus, cowpox, venom; bacillus botulinus, Trail-R1, Trail-R2; cMet, TNF-R family is as LANGF-R; CD27, CD30, CD40; CD95, lymphotoxin a/b receptor, Wsl-1; TL1A/TNFSF15; BAFF, BAFF-R/TNFRSF13C, TRAILR2/TNFRSF10B; TRAILR2/TNFRSF10B; Fas/TNFRSF6CD27/TNFRSF7, DR3/TNFRSF25, HVEM/TNFRSF14; TROY/TNFRSF19; CD40 part/TNFSF5, BCMA/TNFRSF17, CD30/TNFRSF8; LIGHT/TNFSF14; 4-1BB/TNFRSF9, CD40/TNFRSF5, GITR/TNFRSF18; osteoprotegerin/TNFRSF11B; RANK/TNFRSF11A, TRAILR3/TNFRSF10C, TRAIL/TNFSF10; TRANCE/RANKL/TNFSF11; 4-1BB part/TNFSF9, TWEAK/TNFSF12, CD40 part/TNFSF5; Fas part/TNFSF6; RELT/TNFRSF19L, APRIL/TNFSF13, DcR3/TNFRSF6B; TNFRI/TNFRSF1A; TRAILR1/TNFRSF10A, TRAILR4/TNFRSF10D, CD30 part/TNFSF8; GITR part/TNFSF18; TNFSF18, TACI/TNFRSF13B, NGFR/TNFRSF16; OX40 part/TNFSF4; TRAILR2/TNFRSF10B, TRAILR3/TNFRSF10C, TWEAKR/TNFRSF12; BAFF/BLyS/TNFSF13; DR6/TNFRSF21, TNF-α/TNFSF1A, Pro-TNF-α/TNFSF1A; lymphotoxin-beta R/TNFRSF3; lymphotoxin-beta R (LTbR)/Fc chimera, TNFRI/TNFRSF1A, TNF-β/TNFSF1B; PGRP-S; TNFRI/TNFRSF1A, TNFRII/TNFRSF1B, EDA-A2; TNF-α/TNFSF1A; EDAR, XEDAR, TNFRI/TNFRSF1A.

What cherish a special interest is people's target protein of buying with purified form.

Several people's ion channels are the target spots that cherish a special interest.

The example of GPRC includes but not limited to: category-A class rhodopsin receptor, as Musc vertebrates 1 type acetyl group choline, Musc vertebrates 2 type acetyl group choline, Musc vertebrates 3 type acetyl group choline, Musc vertebrates 4 type acetyl group gallbladder alkali; Adrenoceptor (1 type alpha adrenergic receptor, 2 type alpha adrenergic receptors, 1 type beta-2 adrenoceptor, 2 type alpha adrenergic receptors, 3 type alpha adrenergic receptors, 1 type vertebrates dopamine, 2 type vertebrates dopamine, 3 type vertebrates dopamine, 4 type vertebrates dopamine, 1 type histamine, 2 type histamine, 3 type histamine, 4 type histamine, 1 type 5-hydroxy tryptamine, 2 type 5-hydroxy tryptamine, 3 type 5-hydroxy tryptamine, 4 type 5-hydroxy tryptamine, 5 type 5-hydroxy tryptamine, 6 type 5-hydroxy tryptamine, 7 type 5-hydroxy tryptamine, 8 type 5-hydroxy tryptamine, other type 5-hydroxy tryptamine, trace amine, 1 type angiotensin, 2 type angiotensin, bombesin, Kallidin I, the C5a anaphylatoxin, Fmet-leu-phe, class APJ, A type interleukin-8, the Type B interleukin-8, other type interleukin-8,1-11 type C-C chemotactic factor and other type, C-X-C chemotactic factor (2-6 type and other), the C-X3-C chemotactic factor, cholecystokinin CCK, A type CCK, Type B CCK, other CCK, endothelin, (melanocyte stimulates hormone to melanocortin receptor, thyroliberin, the melanocortin receptor hormone), Duffy antigen, prolactin antagonist release peptide (GPR10), neuropeptide tyrosine (1-7 type), neuropeptide tyrosine, other neuropeptide tyrosine, neurotensin, opiates (D, K, M, the X type), somatostatin (1-5 type), tachykinin (substrate P (NK1), substrate K (NK2), neuromedin K (NK3), tachykinin 1, tachykinin 2, vassopressin/oxypressin (1-2 type), oxypressin, oxytocin/mesotocin, cone shell chalone (Conopressin), the class galanin, the albuminoid enzyme activates, appetite peptide and neuropeptide FF, QRFP, the class chemokine receptors, class neuromedin U (neuromedin U, PRXamide), neurophysin (follicle stimulating hormone, progesterone-chorion estrogen, thyrotropin, I type gonadotropin, II type gonadotropin), opsin's (rhodopsin), vertebrates retinal pigment (1-5 type), vertebrates retinal pigment 5 types, the arthropod retinal pigment, arthropod retinal pigment 1 type, arthropod retinal pigment 2 types, arthropod retinal pigment 3 types, the Mollusca retinal pigment, retinal pigment, olfactory sensation (olfactory sensation II fam1-13), prostaglandin (PGE2 EP1 hypotype, PGE2/D2EP2 hypotype, PGE2 EP3 hypotype, PGE2 EP4 hypotype, prostaglandin F2-α, prostacyclin, thromboxane, adenosine 1-3 type, purinoceptor, purinoceptor P2RY1-4,6,11GPR91, purinoceptor P2RY5,8,9,10GPR35,92,174, purinoceptor P2RY12-14GPR87 (UDP-glucose), cannabinoid, platelet activating factor, gonadotropin-releasing hormone, gonadotropin-releasing hormone I type, gonadotropin-releasing hormone II type, class adipokinetic hormone, Corazonin, throtropin releasing hormone and sercretogogue, throtropin releasing hormone, growth hormone cinogenic agent, the class growth hormone cinogenic agent, cast off a skin and trigger hormone (ETHR), melatonin, lysosphingolipids and LPA (EDG), sphingol 1-phosphoric acid Edg-1, lysophosphatidic acid Edg-2, sphingol 1-phosphoric acid Edg-3, lysophosphatidic acid Edg-4, sphingol 1-phosphoric acid Edg-5, sphingol 1-phosphoric acid Edg-6, lysophosphatidic acid Edg-7, sphingol 1-phosphoric acid Edg-8, other leukotriene B42 receptor of Edg, leukotriene B42 receptor BLT1, leukotriene B42 receptor BLT2, category-A orphan/other, infer neurotransmitters, SREB, Mas proto-oncogene relevant with Mas-(MRGs), class GPR45, the cysteinyl leukotriene, G-albumen coupling cholic acid receptor, free-fat acid acceptor (GP40, GP41, GP43), class secretin category-B, calcitonin, corticotropin-releasing factor, gastrin inhibitory polypeptide, glucagon, growth hormone releasing hormone, parathyroid hormone, PACAP, secretin, vasoactive small intestinal polypeptide, Latrophilin, 1 type Latrophilin, 2 type Latrophilin, 3 type Latrophilin, the ETL receptor, brain specificity angiogenesis inhibitors (BAI), class Methuselah (Methuselah) albumen (MTH), cadherin EGFLAG (CELSR), very big g protein coupled receptor, metabotropic glutamate/the pheromone of C class, metabotropic glutamate I-III group, the perception of class calcium, the outer calcium perception of born of the same parents, pheromone, other class calcium perception, infer the pheromone receptor, GABA-B, GABA-B hypotype 1, GABA-B hypotype 2, class GABA-B, orphan GPRC5, orphan GPCR6, there is not seven albumen bridges (Bride of sevenless proteins, BOSS), taste receptors (T1R), the pheromone of D type fungus, class fungus pheromone factors A (STE2, STE3), class fungus pheromone factor B (BAR, BBR, RCB, PRA), E class cAMP receptor, vision albefaction albumen, FZ/level and smooth family, FZ A organizes (Fz1,2,4,5 and 7-9), FZ B organizes (Fz3 and 6), FZ C organizes (other), plough nose receptor, the nematicide chemoattractant receptor, insect odorant receptor and Z class archeobacteria/antibacterial/fungus opsin.

But design motif MURP targeting arbitrary cell albumen includes but not limited to: cell surface protein, secretory protein, cytoplasmic protein and nucleoprotein.0 target spot that cherishes a special interest is an ion channel.

Ion channel is the class protein superfamily, comprises potassium channel (K-passage) family, sodium channel (Na-passage) family, calcium channel (Ca-passage) family, chloride channel (Cl-passage) family and acetyl group choline passage family.Each self-contained subfamily of these families, and each superfamily comprises the special modality that originates from term single gene usually.For example, K-passage family comprises the valtage-gated K-passage subfamily of Kv1.x by name and Kv3.x.Subfamily Kv1.x comprises Kv1.1, Kv1.2 and Kv1.3 passage, and therefore the product of corresponding term single gene is called " kind ".Equally Na-, Ca-, Cl-and other passage family are sorted out.

According to channel operation mechanism ion channel is sorted out.Particularly, the main type of ionophorous protein is characterised in that opening and closing channel protein sees through channel protein and pass the employed method of double-layer of lipoid cell membrane with permission or prevention specific ion.The important channel protein of one class is a voltage-gated channel albumen, and it changes transmembrane potential makes the reaction of opening or closing (door).What cherish a special interest is as the voltage-gated sodium channel 1.6 (Nav1.6) for the treatment of target spot.Another kind of ionophorous protein is a mechanically gated ion channel, and this passage is opened or closed to the mechanical stress on the albumen.Another kind of type is called ligand gated channel, and whether specific ligand and protein binding determine its switching.Part can be the outer part of born of the same parents, as neurotransmitter, or part in the born of the same parents, as ion or nucleotide.

Ion channel allows ion to flow down along the chemical potential gradient is passive usually, yet ionic pump uses the transhipment of ATP antigradient.The coupling transporter that comprises antiporter and co-transport body makes a kind of ionic species antigradient motion by other ionic species energy that motion provides of taking advantage of a situation.

A kind of modal channel protein type that almost can both find at all animal cell membranes allows the potassium ion specificity to pass through cell membrane.Specifically, potassium ion passes through K fast ⁺The channel protein permeates cell membranes is (soon to per second 10 ^-8Ion).And potassium channel protein has differentiates potassium ion and other little alkali metal ion such as Li very accurately ⁺Or Na ⁺Ability.Specifically, potassium ion is bigger 10000 times than the saturating property of sodium ion at least.Potassium channel protein contains four (the same usually) subunits usually, so the target spot of cell surface presents with the tetramer, allows the tetravalence combination of MURP.One class subunit contains six long hydrophobic sections (can be and stride film), and other type comprises two hydrophobic sections.

Another important channel family is a calcium channel.Usually according to electrophysiological characteristics calcium channel is divided into low-voltage and activates (LVA) or high voltage activation (HVA) passage.The HVA passage comprises at least three group passages, promptly known L-, N-and P/Q-type passage.According to its pharmacology and part binding characteristic, these passages are distinguished on electrophysiology and biochemistry.For example, with L-type calcium channel α ₁The bonded dihydropyridine of subunit, diphenyl-alkylamine and piperidines have been blocked a part of HVA calcium current that is called as L-type calcium current in the neuronal tissue.N-type calcium channel is to ω cone shell peptide, but for dihydropyridine compound, flat as nimodipine and nitre benzene ground relative insensitivity.On the other hand, P/Q type passage is insensitive to dihydropyridine, but to infundibulate spider venom Aga IIIA sensitivity.Because big film depolarization activates R-type calcium channel,, therefore classify as high voltage and activate (HVA) passage as L-, N-, P-and Q type passage.R type passage is insensitive to dihydropyridine and ω cone shell peptide usually, but as P/Q, L and N passage to infundibulate spider venom AgaIVA sensitivity.Immunocytochemical stain shows that this receptoroid is positioned at whole brain, especially in the nuclear of dark centerline construction (tail shell nuclear, thalamus, hypothalamus, corpus amygdaloideum, cerebellum) and abdominal part mesencephalic nuclei brain stem.Neuron voltage-sensitive calcium channel is usually by central α ₁Subunit, α ₂/ δ subunit, β subunit and 95kD subunit are formed.

Other non-limitative example comprises Kir (inward rectifyimg potassium channel), Kv (valtage-gated potassium channel), Nav (voltage-gated sodium channel), Cav (valtage-gated calcium channel), CNG (cyclic nucleotide gate passage), HCN (hyperpolarization activate channel), TRP (transient receptor voltage channel), ClC (chloride channel), CFTR (cystic fibrosis transmembrane conductance rate is regulated albumen, chloride channel), IP3R (inositol triphosphate receptor), RYR (Lan Niding receptor).Other channel type is 2-hole path, glutamate receptor (AMPA, NMDA, KA), M2, connection albumen and Cys-ring receptor.

Ionophorous protein is the TMD of 6 following arrangements as the common structure chart of Kv1.2, Kv3.1, Shaker, TRPC1 and TRPC5:

N-end---S1---E1---S2---X1---S3---E2---S4---X2---S5---E3---S6---C-end

Wherein S1-6 is for striding the film sequence, and E1-3 is the cell outer surface ring, and X1-2 is born of the same parents' inner surface ring.E3 ring is the longest and possess hydrophilic property in three born of the same parents' outer shrouds normally, is the bonded good target spot of medicine and MURP.It is poly (as the four poly-or pentamers) complex of striding film α spiral that the hole of multi-channel forms part.Orifice ring is arranged usually, and it is the zone that a proteic wraparound film forms the selectivity filter membrane, has determined which kind of ion to penetrate.This type of passage is called as " orifice ring " passage.

Ion channel is the valuable target spot of drug design, because they relate to physiological process widely.In human body, exist approximately to surpass 300 kinds of ionophorous proteins, wherein many genetic diseasess that relate to.For example, shown that ion channel abnormal expression or dysfunction give rise to diseases, comprised heart, neuron, muscle, respiratory metabolism disease.This part is mainly described ion channel, but same concept and method can be applicable to all memebrane proteins equally, comprises 7TM, 1TM, G-albumen and G-G-protein linked receptor (GPCR) etc.Some ion channels are GPCR.

Ion channel forms the macromolecular complex that comprises the attached protein protomer of combining closely usually, and the multiformity that causes ion channel is used in uniting of this type of subunit.These attached albumen also can with theme MURP, microprotein and toxin targeted integration.

But design motif MURP makes it in conjunction with any passage known in the art and that this paper points out.Can select MURP by any reorganization known in the art or biochemical technology (as expressing and showing) with institute's desired ion passage binding ability (comprising specificity and affinity).For example, can show MURP by the heredity bag that includes but not limited to phage and spore, and carry out the elutriation of anti-intact cell film, or preferred intact cell, as complete mammalian cell.In order to remove and the bonded phage of other non-target cell surface molecular, standard step is low or can't detected similar cell line subdue elutriation to having acceptor levels.Yet Popkov etc. (J.Immunol.Methods 291:137-151 (2004)) show that the relevant cell type is unsatisfactory for subduing, and are significant level because the target spot on their surfaces can reduce usually, and this can reduce the number of required phage clone.Even when the cell of elutriation transfection target spot encoding gene, then to non-transfected cells carry out negative sense select/when subduing, this problem also can take place, particularly when natural target spot gene is not knocked out.Simultaneously, if demonstrations such as Popkov utilize the excessive cell identical with common elutriation (forward selection) to operate, the better effects if of elutriation is selected or subdued to negative sense, unless utilize the antibody of high affinity, target spot specific inhibitor such as micromolecule, peptide or anti-target spot that target spot is sealed, thereby avtive spot can not be utilized.This process is known as " utilize epi-position to cover cell and carry out the negative sense selection ", and this is particularly useful when selecting the theme MURP with desired ion passage binding ability.

In an independent embodiment, the invention provides microprotein, especially the microprotein that at least a ion channel family is had binding ability.The present invention also provides the heredity of showing this type of microprotein bag.The example of the bonded non-limiting ion channel of theme microprotein is sodium, potassium, calcium, acetylcholine and chloride channel.What cherish a special interest is those microproteins and the heredity bag of showing this type of microprotein, and they have binding ability to natural target spot.Natural target spot normally known microorganisms albumen can bonded natural molecule or its fragment and derivant, and it is known to target spot to comprise those that report in the document usually.

The present invention also provides and has showed the heredity bag of the ion channel of modification in conjunction with microprotein.The microprotein of modifying can (a) be compared with corresponding unmodified microprotein, with different ion channel family combinations; (b) compare with corresponding unmodified microprotein, with the different subfamily combinations of same passage family; (c) compare with corresponding unmodified microprotein, combine with the variety classes of same passage subfamily; (d) compare with corresponding unmodified microprotein, combine with the different loci of same passage; And/or (e) compare with corresponding unmodified microprotein, with the same site of same passage in conjunction with but produce different biological effects.

Figure 22 and 46 shows how will be combined into single albumen with bonded microprotein domain of same ion channel different loci or toxin.Bonded two binding sites of these two kinds of microproteins can be from two passages of different families, from two passages of the different subfamilies of same family, from same subfamily but two passages of variety classes (gene outcome) or on two different binding sites on the same passage (kind), perhaps because passage is a polymer, they can (or not simultaneously) be incorporated into the same binding site of same passage (kind) simultaneously.Can be microprotein domain (natural or non-natural, as to contain 2 to 8 disulfide bond), single disulfide bond peptide or linear peptides with bonded binding modules in passage site and domain.Can select these modules independently or with its merging, perhaps can be in the presence of fixed active binding modules select in by the library.Under the latter event, display libraries can be showed multiple module, and wherein a kind of module can comprise the variant library.Typical target is to select to have the more dimer of high-affinity compared with the activated monomer of beginning from the library.

In another embodiment, the invention provides and comprise the albumen of a plurality of ion channels in conjunction with the territory, wherein each domain is modified so that (a) compare with the microprotein domain of corresponding unmodified, this microprotein domain and the combination of different passage family; (b) compare the different subfamily combinations of this microprotein domain and same passage family with the microprotein domain of corresponding unmodified; (c) compare with the microprotein domain of corresponding unmodified, this microprotein domain combines with the variety classes of same subfamily; (d) compare with the microprotein domain of corresponding unmodified, this microprotein domain combines with the different loci of same passage; (e) compare with the microprotein domain of corresponding unmodified, this microprotein domain combines with the same site of same passage but produces the different biological effect; And/or (f) compare with the microprotein domain of corresponding unmodified, the same site of this microprotein domain and same passage also produces identical biological effect.Required is that the microprotein domain can comprise natural or non-natural sequence.Can the single structure territory be coupled together by the allos joint.The single microbial protein structure domain can be in conjunction with identical or different passage family, identical or different passage subfamily, the identical or different kind of same subfamily, the identical or different site of same channels.

The theme microprotein can be toxin.Preferably, toxin moiety or all kept the toxicity spectrum.Specifically, deleterious animal, as Serpentis, when running into some Predators or invador's species, the venom toxin produces different activity to the not isoacceptor of different plant species.Venom has comprised a large amount of relevant or uncorrelated toxin, and each toxin has " activity profile ", and promptly toxin can produce the whole receptors that can measure active whole species.Target spots all in " activity profile " is considered to " natural target spot ", and this comprises people's target spot of the active antagonism of arbitrary toxin.The natural target spot of microprotein or toxin comprises that all documents have reported the quenchable target spot of toxin.Affinity or activity to target spot are high more, and target spot may be natural, primary target spot more, but find that seldom toxin can act on a plurality of target spots of same species.Natural target spot can be the people of the active antagonism of toxin or inhuman receptor.

The back keeps and the toxin of cell binding ability for merging with display carrier, the N-terminal that may need to use synthetic DNA library method test fusions and C-terminal and different position of fusion are (promptly, if the toxin structure territory is for containing cysteine structure territory, in the toxin structure territory before first cysteine or 0,1,2,3,4,5,6 aminoacid after last cysteine), the rich glycine joint library that optimized encoding forms minimum amino acid chain is uncharged, and is most possible compatible with combining of toxin and target spot.Since N-terminal is amino and the C-terminal carboxyl may participate in targeted integration, then this library should comprise the lysine of simulation positively charged amino or the glutamic acid or the aspartic acid (so that merging with toxin C is terminal) of arginine (so that (or) merging with the toxin N-terminal) and the electronegative carboxyl of simulation.

The inhibitor that is used to block target spot when negative sense is selected can be natural or non-natural micromolecule, peptide or albumen.Except simply subduing, the selection of inhibitor mixed thing is the specific useful tool of the designed inhibitors of ion channels of control.Because total total surpass 300 kinds of example passages and have specificity and the sequence similarity that part overlaps, and each passage has a plurality of regulatory sites, has different effect separately, so specific requirement is very complicated.

When modifying the toxin activity maybe when two kinds of different toxin are merged into an albumen, the same site that these two kinds of toxin can be incorporated into same passage has identical physiological effect, or be incorporated into the same site of same passage but have different physiological effecies, or be incorporated into the different loci of same passage, or to be incorporated into the different passages that belong to same subfamily (be Kv1.3 and Kv1.2; Mean different gene outcomes or " kind "), or be incorporated into the different passages (promptly being the K-passage) of identical family, or be incorporated into the passage (being that the K-passage is to the Na-passage) that belongs to different families.

Therefore ion channel has many TMDs (sodium channel has 24) usually, changes the binding site that channel activity provides several differences, non-competing and non-overlapping by different way for instrumentality.A kind of method is created the conjugate in a site on the same channels from the conjugate of the different loci of existence, even these sites have nothing to do.In order to reach this purpose, use the targeting agent of existing toxin, separate by 5,6,7,8,9,10,12,14,16,18,20,25,30,35,40 or 50 amino acid whose variable tap and targeting toxins as targeting 1-2-3-or 4-disulfide bond protein library.When the targeting agent affinity when not being very high this of great use, the affinity in therefore new library has remarkable contribution to overall affinity.Other method is to create new passages regulate thing from the already present instrumentality of the passage of other sequence or structurally associated.For example, conotoxin family comprises the instrumentality of the relevant and structurally associated of the sequence of Ca-, K, Na-passage and nAChR.As if thing is reconciled with the K passage in the library that utilizes the conotoxin derivant, and to be converted to Na passages regulate thing be feasible, vice versa.For example, κ-conotoxin suppresses the K-passage, and mu-conotoxin and Δ-conotoxin suppresses the Na-passage, and omega-conotoxin suppresses the Ca-passage and alpha-conotoxin suppresses acetylcholinergic receptor.

From same ion channel to channel activity have different effect different binding sites utilize variable tap to connect inhibitor to have separately in conjunction with the single inhibitor of two domains of different loci very attractive near making with generation.Or have two single albumen that are incorporated into the domains of the different copies of same loci, an interaction (affinity) that produces the bivalence high-affinity.This method is not used for natural toxin as yet, and is general because their effects rapidly, therefore need keep very little in order to have the maximum tissue permeability, but for pharmacy, speed of action are unimportant, makes it become attracting method.

Therefore can create the library of dimerization, trimerizing, four dimerizations or the multimerization toxin/instrumentality of respectively natural naturally or modification, and directly screen these libraries at protein level, or utilize in these libraries of heredity bag elutriation with affinity (affinity, if take place simultaneously in a plurality of sites in conjunction with) library of improving, then utilize protein expression and purification and based on the experiment of cell, comprise that patch clamp experiments identifies these multimerizations clone's specificity and activity.These individual modules can independent discretely separately elutriation and selection, maybe they can be designed to the common form that exists, therefore the new construction territory is added in the fixed active display systems library that copies that also comprises as library targeting element, and only selects and identify the clone who significantly is better than fixed activated monomer.

Figure 46 and 47 has shown the monomer derived thing that some can be produced by original (natural) toxin, some polymers can with a plurality of different binding site combinations of target spot.Joint is shown as the rPEG of rich glycine, also can utilize molecular library to be optimized but joint can be arbitrary sequence, carries out elutriation then.Utilize above-mentioned multiple mutation strategy to produce the library in the original toxin of activity inside itself, or create the library to enlarge the area that contacts with target spot in the N-terminal or the C-terminal side of active toxin, expectation can be created and other the contacting of target spot.This type of library can be based on the existing toxin that described site is had known activity, and perhaps they can be original 1-, 2-, 3-, the 4-disulfide bond library based on irrelevant microprotein support.These additional contact elements can be added on the one or both sides of active structure domain, and can be directly adjacent with existing adjustment structure territory, or by variable tap and its isolation.Based on domain sequence similarity or polymer domain target spot specificity, initial polymer or final improved polymer can be homology polymer or allos polymer.Therefore, the monomer that comprises this polymer can combine with the identical or different sequence of same target spot.The different natural toxins of known 10-100 kind combine with each passage family, and each clone has 2,3,4,5 or 6 domains, even if only use the natural toxin sequence, also can create and have the multifarious display libraries of huge combination.Can utilize the low-level synthetic mutation based on system's generation replacement rate in amino acid similarity or the family to produce high-quality mutant library, estimate wherein overwhelming majority energy reservation function, it is very big that the function of some characteristics interested improves probability.

Can measure the binding ability of theme MURP, microprotein or toxin and given ion channel according to the Hill coefficient.The stoichiometry of Hill coefficient reflection binding interactions.The Hill coefficient is 2 to show that two kinds of inhibitor are incorporated into each passage.Also can assess allosteric and regulate, this is to be reconciled in conjunction with the activity in a certain site that causes by the far-end site.

Can utilize multiple external and the biologic activity of the interior experimental evaluation ion channel of body or the ability of effect and theme MURP, microprotein or toxin conciliation ion channel activity.For example, can obtain measuring voltage from this area, electric current, transmembrane potential, ion flow such as potassium stream, rubidium stream, ion concentration, gate, the method for second message,second messenger and transcriptional level, and service voltage sensitive dye, radioactive indicator and the electrophysiological method of patch-clamp.Specifically, these experiments can be used for detecting microprotein and the toxin that those can suppress or activate ion channel interested.

Particularly, can compare possible channel inhibitor of detection or activator with appropriate control to measure the conciliation degree.Control sample can be the sample of not handling with candidate's activator or inhibitor.The ion channel activity value that provides compared with the control is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or when lower, exists and suppresses.IC50 is used for determining inhibiting conventional unit (reducing the inhibitor concentration of 50% ion channel activity).IC90 is similarly arranged.Compared with the control, selected given ion channel activity value increases by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500% or more for a long time, realizes that passage activates.

Detect the cell of expression passage interested or the polarization (being current potential) of film and change, with the variation of assessment ion flow.For example, the polar method of a kind of detection cell is to utilize voltage clamp and patch clamp technique, measure electric current with (for example) " cell attaching " pattern, " inside-out " pattern or " full cell " pattern and change (changing) (referring to for example thereby measure polarization, Ackerman etc., New Engl.J.Med.336:1575-1595 (1997)).Utilize standard method can be convenient for measuring full cell currents (referring to for example, Hamil etc., Pflugers.Archiv.391:85 (1981).Other known method comprises: the stream experiment of radioactive label rubidium and utilize voltage sensitive dye fluorescence experiments (referring to for example, Vestergarrd-Bogind etc., J.Membrane Biol.88:67-75 (1988); Daniel etc., J.Pharmacol.Meth.25:185-193 (1991); Holevinsky etc., J.Membrane Biology 137:59-70 (1994)).

Candidate MURP, microprotein or toxin can be measured by the consequence of electric current or ion flow change or electric current or ion flow change for the influence of channel function interested.Candidate albumen may be different to the downstream effect of ion flow.Therefore, can utilize the physiology of any appropriate to change of the influence of evaluation candidate albumen to TCH test channel.The influence of candidate albumen can be by toxin in conjunction with measuring.When utilizing intact cell or zoometry functional outcome, also can measure several effects, as mediator discharge (as dopamine), hormone discharge (as insulin), known or do not identify genetic marker transcribe that variations (as the northern marking), cell volume change (as erythrocyte), immunne response (as t cell activation), cellular metabolism changes as cell growth or pH variation and interior second message,second messenger of born of the same parents such as Ca2 ⁺Variation.

The crucial biologic activity of other ion channel is ion selectivity and gate.Selectivity is the ability that some passage is differentiated ionic species.Allow some ion passing hole channels and get rid of other ions.Gate then is the conversion of open and closed condition.Can be by means known in the art or the open method assessment of this paper.

Other can be used for selecting the biological property of theme MURP, microprotein or toxin is the frequency of target channel switching, is called the gate frequency.The gate frequency is subjected to voltage (in the voltage-gated channel of the switch by the membrane voltage change) and the bonded influence of part.The switching rate of on off state usually＜10 microseconds, but can be increased or reduce by other molecule.Flow velocity (electric current) by hole when ion channel is opened is on per second 10e7 the number of ions magnitude, and the coupling quid pro quo is then few a lot.After the unlatching, some voltage-gated channels enter the non-conduction condition of inactivation, and at this moment they are difficult to carry out depolarization.

Embodiment

Embodiment: design glycine-serine oligomer based on the human sequence

The rich glycine sequence of search in the human genome data base.With three Sequence Identification is suitable donor sequences, sees Table X.

The donor sequences of Table X: GRS design A

Accession number sequence aminoacid protein

NP_009060 GGGSGGGSGSGGGG 486-499 zinc finger protein

Q9Y2X9 GSGSGGGGSGG 19-31 zinc finger protein

CAG38801 SGGGGSGGGSGSG 7-19 MAP2K4

Based on the sequence in the Table X, we have designed and have comprised a plurality of multiple rich glycine sequence of sequence GGGSGSGGGGS peptide A that have.The formation of peptide A oligomerization had formula (GGGSGSGGGGS) _nStructure, wherein n is 2-20.All possible 9 aggressiveness subsequences in the peptide A oligomer that comprises in the listed at least a albumen in Fig. 5 display list 3.Therefore peptide A oligomer does not comprise human T-cell's epi-position.Inspecting the GRS that Fig. 5 discloses based on peptide A oligomer can begin and finish in any site of peptide A.

Embodiment: design glycine-proline oligomer based on the human sequence

Design rich glycine sequence based on sequence GPGGGGGPGGGGGPGGGGPGGGGGGGPGGGGGGPGGG, represent the 146-182 aminoacid of people's 4 class POU domains of accession number NP_006228.Fig. 6 shows that the peptide B oligomer with sequence GGGGGPGGGGP can be used as GRS.POU domain sequence also comprises all and has sequence (GGGGGPGGGGP) _nPeptide in all possible 9 aggressiveness subsequences.Therefore, this class oligomerization sequence does not comprise t cell epitope.

Embodiment: design glycine-glutaminic acid oligomer

Can design rich glycine sequence according to the subsequence GAGGEGGGGEGGGPGG of ribosomal protein S6 kinases (accession number BAD92170) part.For example, the peptide C oligomer with sequence GGGGE forms the sequence that comprises most 9 aggressiveness subsequences in the ribosomal protein S6 kinase sequence.Therefore, has general structure (GGGGE) _nOligomerization GRS have the extremely low risk that comprises t cell epitope.

Embodiment: the rich glycine sequence of surveyor's hydrophilic

The subsequence of the rich glycine residue of search in the human protein data base.These subsequences comprise at least 50% glycine.Only allow to occur among the GRS following non-glycine residue: ADEHKPRST.Identifying 70 kinds of minimum lengths is 20 amino acid whose subsequences.List these subsequences among the adnexa A.Can utilize them to be structured in the GRS that has low immunogen potential in the human body.

Embodiment: make up rPEG_J288

Following embodiment describes the structure coding and contains 288 aminoacid and sequence (GSGGEG) ₄₈The codon optimized gene of URP sequence.At first, we make up filling carrier pCW0051 shown in Figure 40.Figure 42 has shown the expression cassette sequence among the pCWO051.This filling carrier is based on the pET carrier and comprise the T7 promoter.After this vector encoded Flag sequence with the padding sequence in side joint BsaI, BbsI and KpnI site.As shown in figure 42, insert BsaI and BbsI site and therefore after digestion, produce compatibility jag.Behind this padding sequence with His6 label and green fluorescent protein (GFP) gene.This padding sequence comprises termination codon, therefore carries the Bacillus coli cells of filling plasmid pCW0051 and forms no fluorescent colony.Utilize BsaI and KpnI digestion to fill carrier pCW0051.Make up the codon library of the URP sequence of 36 amino acid lengths of coding as shown in figure 41.This URP sequence numbering is rPEG_J36 and has aminoacid sequence (GSGGEG) ₆With the synthetic oligonucleotide of encoding amino acid sequence GSGGEGGSGGEG to and a pair of oligonucleotide annealing of encoded K pnI site adapter form insert.Use following oligonucleotide: pr_LCW0057 forward: AGGTAGTGGWGGWGARGGWGGWTCYGGWGGAGAAGG, pr_LCW0057 is reverse: ACCTCCTTCTCCWCCRGAWCCWCCYTCWCCWCCACT, pr_3KpnI terminator forward: AGGTTCGTCTTCACTCGAGGGTAC, the pr_3KpnI terminator is reverse: CCTCGAGTGAAGACGA.Connect annealed oligonucleotide, obtain representing the product mixtures of the multiple different length of different repetition number rPEG_J12.Utilize sepharose electrophoresis from mixture, to separate the product of corresponding rPEG_J36 length and be connected among the filling carrier pCW0051 of BsaI/KpnI digestion.Most clones induce the back to show green fluorescence in the library of the resulting LCW0057 of being appointed as, and this surperficial sequence rPEG_J36 has been connected in the GFP gene frame.Figure 14 shows the screening of rPEG_J36 sequence and repeats the multimerization process.We have screened the separator that has high-level fluorescence in 288 kinds of separators from the LCW0057 library.48 kinds of separators with hyperfluorescence are carried out pcr analysis confirming the length of rPEG_J section, and identify 16 clones and have required rPEG_J36 length.Obtain the set of 16 kinds of rPEG_J36 separators like this, demonstrate high expressed and have different codons and use.Utilize method shown in Figure 40 that separator is compiled and dimerization.Use BsaI/NcoI digested plasmid mixture, isolate the fragment that comprises rPEG_J36 sequence and a part of GFP.Utilize BbsI/NcoI to digest same plasmid mixture, isolate the fragment that comprises rPEG_J36, most of plasmid vector and residue GFP gene.Two kinds of fragments are mixed, connect and conversion BL21 the fluorescence of screening and separating thing.Carry out two-wheeled dimerization process as shown in figure 14 again.In each was taken turns, we doubled the rPEG_J mrna length, the final gene sets that obtains coding rPEG_J288.The aminoacid of rPEG_J288 and nucleotide sequence are as shown in figure 15.Can see that the rPEG_J288 module comprises the identical but section of the rPEG_J36 that nucleotide sequence is different of aminoacid sequence.Therefore we minimize the inner homology of gene, reduce the risk of spontaneous reorganization.We cultivate the e. coli bl21 that carries coding rPEG_J288 plasmid and have carried out 20 multiplications at least, do not find spontaneous reorganization.

Embodiment: make up rPEG_H288

Utilize the step identical to make up the gene library that coding is called 288 aminoacid URP of rPEG_H288A with making up rPEG_J288.RPEG_H288 has aminoacid sequence (GSGGEGGSGGSG) ₂₄Figure 14 has shown the flow chart of building process.Figure 16 has provided the complete amino acid sequence and the nucleotide sequence of a separator of rPEG_H288.

The serum stability of embodiment: rPEG_J288

The fusion rotein of the URP sequence rPEG_J288 that comprises N-terminal Flag label and merge with A that the green fluorescent protein N-terminal merges was hatched three days in 37 ℃ in 50% mice serum.In the different time points collected specimens, utilize SDS PAGE to analyze and utilize the Western analyzing and testing.The antibody of anti-N-terminal flag label is used for Western and detects.The results are shown in Figure 28, show have 288 amino acid whose URP sequences in serum at least three days be completely stable.

Embodiment: lack the preexisting antibody of rPEG_J288 in the serum

The existence of anti-URP antibody shows and may produce immunogenic response to rich glycine sequence.In order to detect whether there is antibody in the serum, detect the URP-GFP fusions by the ELISA that URP-GFP is fixed in holder and then hatch with 30% serum.Use the existence of detection of anti-IgG horse horseradish peroxidase antibody and substrate and URP-GFP binding antibody.Data as shown in figure 29.Data show, fusion rotein can be arrived by the antibody test of anti-GFP or Flag, but can't be detected by Mus serum.This shows and does not contain the antibody with URP sequence in the Mus serum.

Embodiment: purification comprises the fusion rotein of rPEG_J288

The albumen of our purification structure shape such as Flag-rPEG_J288-H6-GFP.Utilize e. coli bl21 in the SB culture medium, to express this albumen.18 ℃ of following 0.5mM IPTG inducing culture things spend the night.Centrifugal collecting cell.Re-suspended cell agglomerate in containing the TBS buffer of Bezonase and commercially available protease inhibitor cocktail.Heating suspension 10 minutes is with cell lysis in 70 ℃ of water-baths.Centrifugal removal insoluble matter.Utilize fixing metal ions specificity (IMAC) post to handle, then flow through the pillar of fixing resisting-Flag antibody, so that the purification supernatant.Figure 43 shows that the PAGE of purge process analyzes.This process produces the albumen of at least 90% purity.

Embodiment: the fusion rotein that makes up rPEG_J288 and interferon-' alpha '

Utilization is used for the gene of the codon optimized design coding human interferon of escherichia coli expression.With the gene fusion of synthetic gene with coding rPEG_J288.Place N-terminal to be beneficial to detection of fusion proteins and purification on a His6 label.Figure 44 provides the aminoacid sequence of fusion rotein.

Embodiment: make up the rPEG_J288-G-CSF fusions

Utilization is used for the gene of the codon optimized design coding human G-CSF of escherichia coli expression.With the gene fusion of synthetic gene with coding rPEG_J288.Place N-terminal to be beneficial to detection of fusion proteins and purification on a His6 label.Figure 44 provides the aminoacid sequence of fusion rotein.

Embodiment: make up the rPEG_J288-hGH fusions

Utilization is used for the codon optimized design coding human growth hormone's of escherichia coli expression gene.With the gene fusion of synthetic gene with coding rPEG_J288.Place N-terminal to be beneficial to detection of fusion proteins and purification on a His6 label.Figure 44 provides the aminoacid sequence of fusion rotein.

The proteic Expression of Fusion Protein of embodiment: rPEG_J288 and people

RPEG_J288 and two people's albumen-interferon-' alpha 's and human growth hormone's fusion rotein is cloned into T7 expression vector and transformed into escherichia coli BL21.It is 0.5OD that cell grows to optical density at 37 ℃.Then, cell was cultivated 30 minutes at 18 ℃.Then add 0.5mM IPTG and culture is spent the night 18 ℃ of shaken cultivation.Centrifugal collecting cell utilizes BugBuster (Nova base company) (Novagen) to discharge soluble protein, utilizes SDS-PAGE separatin non-soluble albumen and soluble protein composition, utilizes the antibody test fusion rotein of the terminal His6 label of anti-N-by Western.Figure 45 has shown that the Western of two fusion rotein analyzes, and rPEG_J288-GFP in contrast.Fusion rotein all has expression, and major protein is in soluble component.This is the evidence of rPEG_J288 highly dissoluble, because a lot of expressing human interferon-' alpha 's of bibliographical information and human growth hormone's majority attempts all obtaining insoluble occlusion body.Figure 45 has shown the formal representation of most of fusion rotein with full-length proteins, does not promptly detect to show fragment not exclusively synthetic or the Partial Protein degraded.

Polymeric structure of embodiment: VEGF and combination

Make up the library of cysteine restricted peptides as [Scholle, M.D. etc. (2005) Comb Chem HighThroughput Screen, 8:545-51] as described in the document.With anti-people VEGF elutriation is carried out in these libraries, and found two binding modules forming by aminoacid sequence FTCTNHWCPS or FQCTRHWCPI.The oligonucleotide of encoding amino acid sequence FTCTNHWCPS is connected to coding has sequence (GGS) ₁₂The nucleotide sequence of URP sequence rPEG_A36 in.Utilize Restriction Enzyme and Connection Step the fusion sequence dimerization to be comprised the molecule of the 4 copy VEGF binding modules of separating with the rPEG_A36 that is blended in GFP with structure then.Figure 30 has compared the VEGF binding affinity of the fusion rotein that comprises 0-4 VEGF bonding unit.The fusion rotein that only comprises the rPEG_A36 that merges with GFP does not show the affinity to VEGF.Along with the resulting fusion rotein affinity of the increase of VEGF binding modules also increases.

Embodiment: find 1SS binding modules at the treatment target spot

According to [Scholle, M.D. etc. (2005) Comb Chem High ThroughputScreen, 8:545-51] described generation random peptide libraries such as Scholle.Original peptide library has been showed the cysteine restricted peptides with cysteine of being separated by the individual residue at random of 4-10.Following table has been showed the design in library:

Table X: original I SS library:

LNG0001 XXXCXXCXXX X ₃CX ₂CX ₃ NNS?NNS?NNS?TGC?NNS?NNS?TGT?NNS

NNS?NNS

LNG0002 XXCXXXCXXX X ₂CX ₃CX ₃ NNS?NNS?TGC?NNS?NNS?NNS?TGT?NNS

NNS?NNS

LNG0003 XXCXXXXCXX X ₂CX ₄CX ₂ NNS?NNS?TGC?NNS?NNS?NNS?NNS?TGT

NNS?NNS

LNG0004 XCXXXXXCXX X ₁CX ₅CX ₂ NNS?TGC?NNS?NNS?NNS?NNS?NNS?TGT

NNS?NNS

LNG0005 XCXXXXXXCX X ₁CX ₆CX ₁ NNS?TGC?NNS?NNS?NNS?NNS?NNS?NNS

TGT?NNS

LNG0006 CXXXXXXXCX CX ₇CX ₁ TGC?NNS?NNS?NNS?NNS?NNS?NNS?NNS

TGT?NNS

LNG0007 CXXXXXXXXC CX ₈C TGC?NNS?NNS?NNS?NNS?NNS?NNS?NNS

NNS?TGT

LNG0008 CXXXXXXXXXC CX ₉C TGC?NNS?NNS?NNS?NNS?NNS?NNS?NNS

NNS?NNS?TGT

LNG0009 CXXXXXXXXXXC CX ₁₀C TGC?NNS?NNS?NNS?NNS?NNS?NNS?NNS

NNS?NNS?NNS?TGT

LNG0010?XXXXXXCXXCXXXXXX X ₆CX ₂CX ₆ NNS?NNS?NNS?NNS?NNS?NNS?TGC?NNS

NNS?TGTNNS?NNS?NNS?NNS?NNS?NNS

LNG0011?XXXXXCXXXCXXXXXX X ₅CX ₃CX ₆ NNS?NNS?NNS?NNS?NNS?TGC?NNS?NNS

NNS?TGT?NNS?NNS?NNS?NNS?NNS?NNS

LNG0012?XXXXXCXXXXCXXXXX X ₅CX ₄CX ₅ NNS?NNS?NNS?NNS?NNS?TGC?NNS?NNS

NNS?NNS?TGT?NNS?NNS?NNS?NNS?NNS

LNG0013?XXXXCXXXXXCXXXXX X ₄CX ₅CX ₅ NNS?NNS?NNS?NNS?TGC?NNS?NNS?NNS

NNS?NNS?TGT?NNS?NNS?NNS?NNS?NNS

LNG0014?XXXXCXXXXXXCXXXX X ₄CX ₆CX ₄ NNS?NNS?NNS?NNS?TGC?NNS?NNS?NNS

NNS?NNS?NNS?TGT?NNS?NNS?NNS?NNS

LNG0015?XXXCXXXXXXXCXXXX X ₃CX ₇CX ₄ NNS?NNS?NNS?TGC?NNS?NNS?NNS?NNS

NNS?NNS?NNS?TGT?NNS?NNS?NNS?NNS

LNG0016?XXXCXXXXXXXXCXXX X ₃CX ₈CX ₃ NNS?NNS?NNS?TGC?NNS?NNS?NNS?NNS

NNS?NNS?NNS?NNS?TGT?NNS?NNS?NNS

LNG0017?XXCXXXXXXXXXCXXX X ₂CX ₉CX ₃ NNS?NNS?TGC?NNS?NNS?NNS?NNS?NNS

NNS?NNS?NNS?NNS?TGT?NNS?NNS?NNS

LNG0018?XXCXXXXXXXXXXCXX X ₂CX ₁₀CX ₂ NNS?NNS?TGC?NNS?NNS?NNS?NNS?NNS

NNS?NNS?NNS?NNS?NNS?TGT?NNS?NNS

Use following proposal at the relevant target spot elutriation library of a series of treatment: to utilize to contain the antigenic PBS4 of 5 μ g/ml target spots ℃ bag and spent the night by immunoadsorption elisa plate hole.By plate, seal non-specific site 2h with PBS flushing bag with sealing buffer (PBS that contains 0.5%BSA or 0.5% ovalbumin) room temperature.Use PBST (PBS that contains 0.05% polysorbas20) flushing plate then, in the hole, add and contain 1-5x10 ¹²The binding buffer liquid of/ml phage particle (the sealing buffer that contains 0.05% polysorbas20), 2h vibrates under the room temperature.Empty Kong Bingyong PBST flushing.Utilize and hatch 10 minutes elution of bound phagies from the hole under the 100mM HCl room temperature, transfer in the sterile tube and utilize 1M TRIS alkali neutralization.In order to infect, in neutral phage eluate, be added among the logarithmic (log) phase escherichia coli SS320 that the Super meat soup that contains 5 μ g/ml tetracyclines cultivates, and in 37 ℃ of shaken cultivation 30 minutes.Then the culture that infects is transferred in the bigger pipe that the Super meat soup that contains 5 μ g/ml tetracyclines is housed, 37 ℃ of shaken cultivation are spent the night.Centrifugal overnight culture is removed escherichia coli, and adds 20%PEG and 2.5M NaCl solution to PEG concentration 4%, makes the phage particle precipitation.Centrifugal collecting precipitation phage, and the phage agglomerate is resuspended among the 1ml PBS is centrifugally removed remaining escherichia coli and is also transferred in the new pipe.Spectrophotography estimation phage concentration is used phage to carry out next round and is selected.3 or 4 take turns the phage elutriation after, screen the binding affinity of single clone to target spot.Select the single plaque that is selected from phage clone in the middle of the elutriation and be transferred to and contain in the 5 μ g/ml tetracycline Super meat soups, and spend the night 37 ℃ of shaken cultivation.4 ℃ are spent the night by elisa plate with 3 μ g/ml antigens and reference protein (BSA, ovalbumin, IgG) bag.By plate, seal non-specific site (PBS that contains 0.5%BSA or 0.5% ovalbumin) 2h with PBS flushing bag with sealing buffer room temperature.Escherichia coli in the centrifugal removal incubated overnight liquid, with binding buffer liquid (the sealing buffer that contains 0.05% polysorbas20) with 1:10 dilution supernatant and transfer in the elisa plate that PBST (PBS that contains 0.05% polysorbas20) washed.Plate 2h is hatched in vibration under the room temperature.After the PBST flushing, in the hole, add with PBS with (Pharmacia) antibody of anti--M13-HRP (Pharmacia Corporation) of 1:5000 dilution.Vibration was hatched this plate 30 minutes under the room temperature, and utilized PBST and PBS elder generation afterflush.The 0.4mg/ml ABTS and the 0.001%H that will contain the preparation of 50mM phosphoric acid sodium citrate buffer solution ₂O ₂Substrate solution join in the hole, develop the color after 40 minutes, read plate at 405nm.This ELISA reading can be determined clone-specific, and can utilize ripe commercialization method that the antigenic specificity clone is checked order.

Table X: the sequence of EpCAM specificity binding modules

S Y I C H N C L L S sNG0017S3.021

L R C W G M L C Y A sNG0017S3.017

L R C I G Q I C W R sNG0017S3.022

L K C L Y N I C W V sNG0017S3.024

R P G M A C S G Q L C W L N S P sNG0018S3.015

P H A L Q C Y G S L C W P S H L sNG0018S3.018

R A G I T C H G H L C W P I T D sNG0018S3.019

R P A L K C I G T L C S L A N P sNG0018S3.014

P H G L W C H G S L C H Y P L A sNG0018S3.012

P H G L I C A G S I C F W P P P sNG0018S3.007

P R N L T C Y G Q I C F Q S Q H sNG0018S3.011

P H N L A C Q N S I C V R L P R sNG0018S3.021

P H G L T C T N Q I C F Y G N T sNG0018S3.006

L F C W G N V C H F sNG0017S3.006

L T C W G Q V C F R sNG0017S3.009

R C P S R V P W C V sNG0017S3.011

Q L V C G F S D S S R L C Y M R sNG0018S3.009

L L C Y I T S P G N R L C S P Y sNG0018S3.022

Table X: the sequence of VEGF specificity binding modules

W E C T Q H W C P S sNG0025S3.021

A P F F S C S F G F C R D L Q T sNG0026S3.035

T P Y F R C Q F G F C F D S F S sNG0026S3.045

N P F F Y C V A G K C V D A P L sNG0026S3.029

D M R F L C R H G K C H D L P L sNG0026S3.034

P P F F V C S L G K C R D A H L sNG0026S3.043

P P Q F Q C V R G K C F D L T F sNG0026S3.053

I S T F F C S N G S C V D V P A sNG0026S3.006

P P H F R C F N G S C V D L S R sNG0026S3.051

N V H F W C H N H K C H D L V S sNG0026S3.040

L F F K C D V G H G C Y D I K H sNG0026S3.038

L Y F Q C F P N R G C S T L Q P sNG0026S3.002

P S F F C S P?L L G C R D S L S sNG0026S3.052

G T P R C N P F R Q F C A I P S sNG0026S3.032

L C L P L G R W C P sNG0025S3.016

T S P A C N P F R H F C T L P T sNG0026S3.058

Q P P I C N P F R Q L C G I P L sNG0026S3.046

V H T F C N P F R Q M C S L P M sNG0026S3.027

R M V N C N P F N S W C S L P S sNG0026S3.001

S K H M C N P F H S W C G V P L sNG0026S3.047

R W P V C N P F L G Y C G I P N sNG0026S3.056

S K P T C N V F N S W C S V P L sNG0026S3.059

R P P A C N L F L S W C S Y D S sNG0026S3.004

G R S V C N P Y K S W C P V R Q sNG0026S3.011

A S S C K D S P H F R C L F P L sNG0026S3.055

L A N C P N S P G F L C L H A V sNG0026S3.024

P F A C P H S S G F R C L Y N I sNG0026S3.005

S F T C S L F P S P H C T T L R sNG0026S3.054

L R L C T Y G G G K Y D C S S T sNG0026S3.050

G S Y C Q Y R P F S S F C N R S sNG0026S3.048

C S Y N Q V L G R A C sNG0025S3.001

P H C R Q H P L D R W M C S P S sNG0026S3.057

S L C S M F G?D T P H W N C V P sNG0026S3.007

S S C S L F N N T R H W S C T D sNG0026S3.008

Table X: the sequence of CD28 specificity binding modules

T T A Y P D C F W C S L F G P P sNG0028S3.085

M L D T T I C P W C S L F G P V sNG0028S3.081

M L X T T I C P W C S L F G P V sNG0028S3.018

E L L L E R C S W C S L F G P P sNG0028S3.086

S L S Q Q S C D W C W L F G P P sNG0028S3.060

K R L L E C G A L C A L F G P P sNG0028S3.008

H T I L T C D S G F C T L F G P sNG0028S3.012

N L W H V C H T S L C H S R L A sNG0028S3.092

N S F Y L C H S S V C G Q L P S sNG0028S3.082

A G F S C E N Y F F C P P K N L sNG0028S3.016

S W C T V F G N H D P S C N S R sNG0028S3.004

C S S N G R W K A H C sNG0028S3.076

L P N M W R V V V P D V Y D R R sNG0028S3.068

Table X: the sequence of CD28 specificity binding modules

K H Y C F G P K S W T T C A R G sNG0030S3.096

P W C H L C P G S P S R C C Q P sNG0030S3.091

P E S K L I S E E D L N G D V S sNG0030S3.042

Table X: the sequence of Tiel specificity binding modules

I W D R V C R M N T C H Q H S H sNG0032S3.096

P Y T I F C L H S S C R S S S S sNG0032S3.087

D W C L T G P N T L S F C P R R sNG0032S3.031

Table X: the sequence of DR4 specificity binding modules

L S T W R C L H D V C W P P L K sNG0033S3.072

Table X: the sequence of DR5 specificity binding modules

V Y L T Q C G A Q L C L K R T N sNG0034S3.039

P Y L T S C G D R V C L K R P P sNG0034S3.001

P Y L S R C G G R I C M H D R L sNG0034S3.026

L K L T P C S H G V C M H R L R sNG0034S3.087

Y Y L T N C P K G H C L R R V D sNG0034S3.080

L Y L H S C S R G I C L S P R V sNG0034S3.082

F S C Q S S F P G R R M C E L R sNG0034S3.040

H R C S A H G S S S S F C P G S sNG0034S3.029

Table X: the sequence of TrkA specificity binding modules

K T W D C R N S G H C V I T F K sNG0035S3.074

A T W D C R D H N F S C V R L S sNG0035S3.089

Embodiment: aEpCAM drug conjugates

From the random peptide library that produces according to [Scholle, M.D etc. (2005) Comb Chem High ThroughputScreen, 8:545-51] such as Scholle, separate anti--EpCAM peptide.Original peptide library has been showed the cysteine restricted peptides with cysteine of being separated by the individual residue at random of 4-10.After above-mentioned library carried out the affine selection of three-wheel, be separated to several EpCAM specific peptide parts (EpCam1 (Table X).EpCam1 has four amino acid whose conservative cysteine (CXXXXC) at interval from thing.The codon that utilizes 3-9 residue of coding is EpCam1 part soft (softly) randomization (except the cysteine position), and moves to the phasmid carrier.Then with EpCAM to the phasmid library carry out affinity select with separation help bonded peptide part (Table X, EpCam2).The EpCam2 part comprises conservative CXXXXC cysteine sept.In addition, most resisting-EpCam sequence does not contain lysine residue, helps coupling free amino outside binding sequence like this.And, can resist usually-EpCam peptide part and the fusion of (random length) URP sequence, and utilize the repetition dimerization to carry out multimerization.Can use gained to resist-the selectively targeted EpCAM of EpCAM MURP, its affinity is higher than sequence monomer.Figure 31 shows the example of a tetramer EpCAM-URP aminoacid sequence.This sequence only comprises two lysine residues that are positioned at N-terminal Flag label.The side chain of these lysine residues is especially suitable for drug coupling.

Table X. anti--the EpCam sequence

The title sequence

EpCam1 RCWGMLCYA

LRCIGQICWR

L KCLYNICWV

LFCWGNVCHF

LTCWGQVCFR

RPGMACSGQLCWLNSP

PHALQCYGSLCWPSHL

RAGITCHGHLCWPITD

RPAL KIGTLCSLANP

PHGLWCHGSLCHYPLA

PHGLICAGSICFWPPP

PRNLTCYGQICFQSQH

PHNLAC?QNSICVRLPR

PHGLTCTNQICFYGNT

EpCam2 HSLTCYGQICWVSNI

PTLTCYNQVCWVNRT

PALRCLGQLCWVTPT

PGLRCLGTLCWVPNR

RNLTCWNTVCYAYPN

RGL KCLGQLCWVSSN

PTL KCSGQICWVPPP

RNLECLGNVCSLLNQ

PTLTCLNNLCWVPPQ

RGL KCSGHLCWVTPQ

HGLTCHNTVCWVHHP

HTLECLGNICWVINQ

HGLTCYNQICWAPRP

HGLACYNQLCWVNPH

RGLACQGNICWRLNP

RAITCLGTLCWPTSP

LTLECIGNICYVPHH

Embodiment: add random sequence

By at N-terminal, C-terminal or the N of binding sequence with C-terminal adds top connection and random sequence can make affine maturation of binding modules or prolongation.Figure 32 demonstration is added in the restricted sequence of original cysteine on anti--EpCAM binding modules.Can use strand or double-stranded DNA cloning process to produce and add the random sequence library.In case produce, the available initial target protein or second albumen carry out affine selection to the library.For example, comprise the anti--interpolation library of EpCAM binding modules and can be used for selecting to comprise the sequence of two or more target protein binding sites.

Embodiment: make up 2SS and make up the library

Design a series of oligonucleotide to make up based on the library of VEGF in conjunction with 1SS peptide FTCTNHWCPS.In the side joint sequence of oligonucleotide, mix variation with the cysteine distance mode, but the VEGF peptide binding sequence is maintained fixed.

The forward oligonucleotide:

LMS70-1

CAGGCAGCGGGCCCGTCTGGCCCGTGYTTTAC TTGTACGAATCATTGGTGTCCT

LMS70-2

CAGGCAGCGGGCCCGTCTGGCCCGTGYNNKTTTAC TTGTACGAATCATTGGTG TCCT

LMS70-3

CAGGCAGCGGGCCCGTCTGGCCCGTGYNNKNNKTTTAC TTGTACGAATCATTG GTGTCCT

LMS70-4

CAGGCAGCGGGCCCGTCTGGCCCGTGYNHTNHTNHTTTTAC TTGTACGAATCA TTGGTGTCCT

LMS70-5

CAGGCAGCGGGCCCGTCTGGCCCGTGYNHTNHTNHTNHTTTTAC TTGTACGAA TCATTGGTGTCCT

LMS70-6

CAGGCAGCGGGCCCGTCTGGCCCGTGYKMTKMTKMTKMTKMTTTTAC TTGTA CGAATCATTGGTGTCC

Reverse oligonucleotide (reverse complemental):

LMS70-1R

ACCGGAACCACCAGACTGGCCRCACGAAGGACACCAATGATTCGTACAA

LMS70-2R

ACCGGAACCACCAGACTGGCCRCAMNCGAAGGACACCAATGATTCGTACAA

LMS70-3R

ACCGGAACCACCAGACTGGCCRCAMNNMNNCGAAGGACACCAATGATTCGTACAA

LMS70-4R

ACCGGAACCACCAGACTGGCCRCAADNADNADNCGAAGGACACCAATGATTCGTACAA

LMS70-5R

ACCGGAACCACCAGACTGGCCRCAADNADNADNADNCGAAGGACACCAATGATTCGTACAA

LMS70-6R

ACCGGAACCACCAGACTGGCCRCAAKMAKMAKMAKMAKMCGAAGGACACCAATGATTCGTACAA

Oligonucleotide solution

Mixture 1 (from 100 μ M mother solutions): 100 μ l70-6,33 μ l70-5,11 μ l70-4,3.66 μ l70-3,1.2 μ l70-2,0.4 μ l70-1.Mixture 2 (from 100 μ M mother solutions): 100 μ l70-6R, 33 μ l70-5R, 11 μ l70-4R, 3.66 μ l70-3R, 1.2 μ l70-2R, 0.4 μ l70-1R

The PCR assembling

10.0 μ l template oligonucleotide (5 μ M), 10.0 μ l10X buffer, 2.0dNTPs (10mM), 1.0 μ l cDNA polymerases (clone Imtech) (Clonetech), 77 μ l DS H ₂O.The PCR program: 95 ℃ 1 minute, (95 ℃ 15 seconds, 54 ℃ 30 seconds, 68 ℃ 15 seconds) x5,68 ℃ 1 minute.

Pcr amplification

Primer, 10.0 μ l assembling mixture, 10.0 μ l10X buffer, 2.0dNTPs (10mM), 10.0 μ lLIBPTF (5 μ M), 10.0 μ l LIBPTR (5 μ M), 1.0 μ l cDNA polymerases (clone Tyke), 57 μ l DSH ₂O.The PCR program: 95 ℃ 1 minute, (95 ℃ 15 seconds, 54 ℃ 30 seconds, 68 ℃ 15 seconds) x25,68 ℃ 1 minute.Utilize Amican post Y10 purified product.Utilize SfiI and BstXI digestion product and connect into phasmid carrier pMP003.16 ℃ of connections are spent the night on MJ PCR instrument.The EtOH deposition and purification connects product.Use electroporation to be transformed in the fresh competence ER2738 cell.

As described belowly elutriation is carried out in the gained library with VEGF.Identify the separator that several relative 1SS homing sequence VEGF binding abilities improve.Figure 38 has shown combination and expression data.Figure 39 has shown sequence and has made up clone's Western analytical data.

Embodiment: make up the phage elutriation in library

First round elutriation:

1) first round, every library bag is screened by 4 holes.Utilization contains 0.25 μ g VEGF ₁₂₁Antigenic 25 μ lPBS bag is reached (Costar) 96-hole elisa plate by Coase.With shrouding film shrouding.Can 4 ℃ of bags spent the night or at 37 ℃ of bags by 1 hour.

2) throw away bag by solution after, add 150 μ l PBS/BSA1% blind holes.Shrouding was also hatched 1 hour at 37 ℃.

3) throw away lock solution after, in the hole, add the phage (referring to the library scheme that increases again) of 50 μ l prepared fresh.Be only applicable to the first round: the tween that also adds 5 μ l5%.Shrouding was also hatched 2 hours at 37 ℃.。

Simultaneously, 2ml SB culture medium is added that 2 μ l5mg/ml tetracyclines and 2 μ l ER2738 cellular preparations hatch altogether, 37 ℃ of 250rpm growth 2.5h.A culture is cultivated in each screening library, comprises the negativity selection.Take all measures to be polluted with the culture of avoiding containing phage.

4) throw away phage solution, every hole adds 150 μ l PBS/ tweens 0.5% and violent piping and druming 5 times.After 5 minutes, throw away and repeat rinsing step.In the first round, wash like this 5 times, second takes turns 10 times, and the 3rd, 4,5 take turns 15 times.

5) throw away final rinse solution after, add the tryptic PBS of 10mg/ml contain 50 μ l prepared fresh, shrouding was also hatched 30 minutes at 37 ℃.Violent piping and druming 10 times, (first round 4x50 μ l, second takes turns 2x50ml, subsequent rounds 1x50 μ l) transfers in the 2ml culture of Escherichia coli of preparation with eluate, and at room temperature hatches 15 minutes.

6) SB culture medium, 1.6 μ l Carbenicillins and the 6 μ l5mg/ml tetracyclines of adding 6ml preheating.Culture is transferred in the 50ml polypropylene tube.

7) 37 ℃ of 250rpm vibration 8ml culture 1h add 2.4 μ l100mg/ml Carbenicillins, again in 37 ℃ of 250rpm vibration 1h.

8) add 1ml VCSM13 helper phage and transferring in the 500ml polypropylene centrifuge bottle.(37 ℃) the SB culture medium and 46 μ l100mg/ml Carbenicillins and the 92 μ l5mg/ml tetracyclines that add the 91ml preheating.37 ℃ of 300rpm vibration 100ml culture 1/2-2h.

9) add 140 μ l50mg/ml kanamycin and continue 37 ℃ of 300rpm shaken overnight.

10) 4 ℃ of 4000rpm centrifugal 15 minutes.Transfer to supernatant in the clean 500ml centrifuge bottle and add 25ml20%PEG-8000/NaCl2.5M.Placed on ice 30 minutes.

11) 4 ℃ of 9000rpm centrifugal 15 minutes.Supernatant discarded, napkin are inverted to put and are done at least, and wipe remaining liq with napkin from centrifuge bottle top.

12) blow and beat up and down along centrifuge bottle one side the phage agglomerate is resuspended in 2ml PBS/BSA0.5%/tween 0.5% buffer.It is resuspended to utilize the 1ml pipet further to blow and beat up and down, centrifugal 1 minute of 4 ℃ of full speed, and supernatant is by flowing into clean 2ml centrifuge tube behind the 0.2 μ m filter membrane.

13) connect step 3) and carry out next round, and the phage prepared product is stored in 4 ℃.Add 0.02% (w/v) Hydrazoic acid,sodium salt and can do long term storage.Every phage of only using prepared fresh of taking turns.

Second takes turns elutriation

Second takes turns, and every library bag is screened by 2 holes.Utilization contains 0.25 μ g VEGF ₁₂₁Antigenic 25 μ lPBS bag is reached (Costar) 96-hole elisa plate by Coase.With shrouding film shrouding.Can 4 ℃ of bags spent the night or at 37 ℃ of bags by 1 hour.

Each library is also sealed 2 and is not wrapped by the hole as the negative control that calculates accumulation rate.

The third round elutriation

Third round, every library bag is screened by 1 hole.Utilization contains 0.25 μ g VEGF ₁₂₁Antigenic 25 μ lPBS bag is reached (Costar) 96-hole elisa plate by Coase.With shrouding film shrouding.Can 4 ℃ of bags spent the night or at 37 ℃ of bags by 1 hour.

Each library is also sealed 1 and is not wrapped by the hole as the negative control that calculates accumulation rate.

Embodiment: based on the elutriation of solution:

1. illustrate the target protein biotinylation according to the manufacturer.

2. spent the night by 8 holes (each selection) altogether and in 4 ℃ of bags with containing 1.0 μ g neutravidin (Pierre Si company) PBS bag (Pierce).

3. used SuperBlock (Pierre Si company) blind hole under the room temperature 1 hour.To comprise the plate that seals buffer and store stand-by (in the step 6).

4. use 100nM biotinylation target protein and add 1012 phagies/ml (being dissolved among the PBST), use SuperBlock and 0.05% polysorbas20 to make cumulative volume be 100-200 μ l.

5. suspendible phage-target spot mixture 1h at least overturns under the room temperature.

6. utilize 700 μ l SuperBlock to dilute 100 μ l phage-target spots, mixed and 8 neutravidin (neutravidin) bag by plate hole in every hole add 100 μ l (from step 3).

7. hatched under the room temperature 5 minutes.

8. with PBST flushing 8 times.

9. with 100 μ l100mM HCl wash-out bacteriophages 10 minutes.

10. add 10 μ l1M TRIS pH=8.0 neutralization.

11. infect the cell that is used for bed board, the amplification phage is used for the next solution elutriation of round.

Embodiment: with phage E LISA screening VEGF positive colony

1) in 96 hole depth orifice plates, adds the 0.5ml SB that contains 50 μ g/ml Carbenicillins.Select a clone and incubated cell.

2) 37 ℃ of 300rmp vibration culture plate of containing bacterial cultures spends the night.

3) the PBS solution of preparation 4ng/ μ l target point protein.Add 25 μ l (100ng) albumen in every hole and 4 ℃ of overnight incubation.

4) throw away the elisa plate of bag quilt and with PBS flushing 2 times.Add 150 μ l/ hole PBS+0.5%BSA (sealing buffer).Seal 1h under the room temperature.

5) centrifugal miniature pipe support (3000rpm; 20 minutes).

6) preparation binding buffer liquid (sealing buffer+0.5% polysorbas20).The every hole of packing 135 μ l binding buffer liquid in low protein binding 96 orifice plates.

7) dry the elisa plate hole also with PBST (PBS+0.5% polysorbas20) washing 2 times.

8) with PBST dilution 15 μ l phagies from overnight culture, the piping and druming mixing also shifts 30 μ l at each albumen bag in by the hole.Gentle vibration 2h under the room temperature.

9) wash plate 6 times with PBST.

10) every hole adds the 50 μ l binding buffer liquid that contain the M13-HRP1:5000 dilution.Gentle vibration is 30 minutes under the room temperature.

11) wash plate 4 times with PBST, use H ₂O washes plate 2 times.

12) (the 5.88ml sodium citrate buffer solution adds 120 μ l ABTS and 2 μ lH to preparation 6ml ABTS solution ₂O ₂).Every hole packing 50 μ l of each elisa plate.

13) hatch under the room temperature and with the ELISA plate reading machine according to signal when appropriate between point (1h at the most) read 405nm O.D..

Embodiment: the dimerization of binding modules

Create the phage display library of 10e9-10e11 kind cyclic peptide according to standard method, cyclic peptide comprises the aminoacid of 4,5,6,7,8,9,10,11 and 12 randomizations or incomplete randomization between the disulfide bond cysteine, and at cysteine the outside is had other randomization aminoacid under the certain situation.Comprise these cyclic peptide libraries of people VEGF elutriation with many target spots, produce reliably that specificity is incorporated into hVEGF but not in conjunction with the peptide of BSA, ovalbumin or IgG.

Embodiment: structure and elutriation are based on the library of plexin

According to two libraries of Plexin support Design.Use the Pfam albumen database that the plexin domain of natural generation is carried out the phylogenetics comparison as shown in figure 35.Intersection region when N-and the generation of C-library are guarded and be used as to plexin support mid portion (Cys24-Gly25-Trp26-Cys27) in two library designs.Figure 36 shows the randoming scheme in two plexin libraries.The oligonucleotide in two libraries of coding is overlapped on the intersection region, and behind pull-thru PCR, carry out restricted clone (SfiI/BstXI), be cloned among the phasmid carrier pMP003, thereby obtain two libraries.Gained plexin library is called as LMP031 (N-terminal library) and LMP032 (C-terminal library), and complexity separately is about 5x108 independent transformant.By PCR to each not about 24 Carb resistance clones in selection storehouse analyze with the checking.The clone who produces correct big or small fragment (375bp) is further carried out the dna sequencing analysis.From LMP031 and LMP032 library, obtain the plexin sequence of 50% and 67% correct total length respectively.

Ratio with 50/50 is carried out parallel elutriation with the mixed VEGF, death receptor Dr4, ErbB2 and the HGFR that are fixed on the 96 hole elisa plates of using in two libraries.Carry out 4 and take turns elutriation, the first round is used 1000ng albumen, and second takes turns use 500ng albumen, and third round is used 250ng albumen, and four-wheel uses 100ng albumen.After last takes turns elutriation, analyze combining of each 192 Carb resistance clones selecting and 100ng ankyrin target spot, human IgG, ovalbumin and BSA with anti--M13 polyclone Ab of phage E LISA and coupling horseradish peroxidase.The positive colony ratio of the following target spot that obtains is the highest: be followed successively by DR4 (69%), ErbB2 (53%), HGFR (13%) and BoNT target spot (1%).Positive colony is further carried out PCR and dna sequencing analysis.All clones disclose unique sequences, except a clone (at DR4) derived from LMP032 (C-terminal library).Figure 37 has shown the target spot Selective Separation thing sequence of some evaluations.

For further analysis, at first that a class is selected target spot specificity junction mixture sub-clone with the form production of solubility microprotein, finally carries out purification with pyrolysis method then in protein expression carrier pVS001.Target spot specific microbial proteins to purification carries out the protein elisa assay to confirm target spot identification, and SDS-PAGE confirmation form body forms, and surperficial plasmon resonance detects the affinity with target spot.Best clone is used for the lower whorl library to produce with its characteristic of further improvement.

Embodiment: make up library based on snake venom

Use standard method to create the phage display library that 10e8-10e10 three refers to toxin (3FT) supports, its middle finger 1 and refer to 2 sloping portion or refer to 3 and refer to that 2 rising part contains incomplete randomization aminoacid.

Use two 3FT supports as the template that produces the 3FT library (refer to 1 and refer to 2 structures).Figure 33 shows the structure of 3FT support and the multiple sequence comparison of correlated series.Design two surface ring randomizations that the library makes toxin as shown in figure 34.The oligonucleotide in four libraries of coding is overlapped on the annealing region, and carry out pull-thru PCR, follow restricted clone (SfiI/BstXI) in phage vector pMP003, obtain the 3FT support library of incomplete randomization.The 3FT library of gained is called LMP041.

Embodiment: with the binding peptide grafting in the microprotein support-the auxiliary randomization of target spot specific peptide

Here purpose is to use and is identified that target spot interested is had specific peptide generation 3SS adds the target spot specificity junction mixture.This strategy uses the VEGF specific peptide to transfer in the finger 1 of 3FT support, and modifies and refer to 2 AA residue, and this residue and target spot specific sequence are approaching, with the VEGF conjugate of generation high-affinity.Utilize standard method as mentioned above to create the phage display library of 10e8-10e10 3 finger toxin (3FT) supports, wherein contain and refer to 1 VEGF specific sequence and refer to 2 part sloping portion at random, refer to that at random the be encoded F1-VEGF specificity forward primer of following sequence of 1 forward primer substitutes: PS G P S C H T T N H W P I S A V T C P P except 2.

The oligonucleotide in four libraries of coding is overlapped on the annealing region and carries out pull-thru PCR, restricted then clone (SfiI/BstXI) is in phage vector pMP003, and the incomplete randomization that contains that obtains paying close attention to refers to (VEGF specificity) 3FT support library of 2.The 3FT library of gained is called LMP042.

The plasma half-life of embodiment: MURP

Basic as [Pepinsky, R.B. etc. (2001) JPharmacolExp Ther, 297:1059-66] described plasma half-life of measuring MURP after with MURP i.v. or i.p. infusion cannula rat.At different time points (5 minutes, 15 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 1 day, 2 days, 3 days) blood sampling and utilize ELISA to detect the plasma concentration of MURP.(Scientific Consulting Inc., Apex NC) calculate pharmacokinetic parameter to utilize WinNonlin2.0 version (scientific adviser company).In order to analyze the effect of URP, can relatively contain the proteic plasma half-life of URP module and not contain the plasma half-life of the same protein of URP module.

Embodiment: MURP's melts property testing

To be in purification MURP sample concentration in physiological buffer such as the phosphate buffered saline (PBS) to the variable concentrations of scope between 0.01mg/ml-10mg/ml, to measure the dissolubility of MURP.Sample can be hatched and grow to several weeks.Muddy precipitation appears in the sample that concentration surpasses the MURP dissolubility, can pass through spectrophotometric determination.Can remove deposit by centrifugal or filtration, utilize the albumen experiment as Bu Ladefu (Bradford) measuring 280nm absorbance then, to detect protein concentration in the supernatant.Can melt again with the accelerate dissolution degree at-20 ℃ of freezing samples and study.This process often causes poorly soluble albumen precipitation.

The serum of embodiment: MURP is in conjunction with activity

Available interested MURP bag is by microwell plate, with the reference protein bag by other hole in the plate.Then, in the hole, add interested serum sample and hatch 1h.Then, with washing plate machine washing plate.What bonded serum albumin can be added into detects with enzyme such as horseradish peroxidase or the link coupled antiserum protein antibodies of alkali phosphatase.Detect the bonded another way of MURP serum and be and add interested MURP about 1h to the serum and make its combination.Then, the antibody mediated immunity with epi-position in the anti-MURP sequence precipitates MURP.Sedimentary sample is carried out PAGE analyze, optional albumen with Western analyzing and testing and MURP co-precipitation.Utilize mass spectrum can detect the serum albumin of co-precipitation.

Claims (28)

1. a unstructured recombinant polymers (URP), it substantially can not the non-specific binding serum albumin, it is characterized in that:

(a) contain at least 100 contiguous amino acids;

(b) contained glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residue sum accounts among the described URP all amino acid whose about more than 80% among the described URP;

(c) measure through the Chou-Fasman algorithm, at least 50% aminoacid of described URP sequence does not form secondary structure;

(d) scoring of the T epi-position of described URP is less than-4.

2. URP as claimed in claim 1 is characterized in that described URP comprises glycine.

3. URP as claimed in claim 1 is characterized in that described URP comprises glutamic acid.

4. as each described URP among the claim 1-3, it is characterized in that described URP comprises the alpha-non-natural amino acid sequence.

5. as each described URP among the claim 1-3, it is characterized in that, select described URP to mix in the heterologous protein, described URP is mixed heterologous protein after, compare with the respective egg white matter that does not contain described URP, the serum half-life of described heterologous protein is long and/or dissolubility is higher.

6. as each described URP among the claim 1-3, it is characterized in that described aminoacid mainly is the hydrophilic residue.

7. as each described URP among the claim 1-3, it is characterized in that the aminoacid that is selected from any type of glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) or proline (P) accounts among the described URP all amino acid whose about more than 20% separately.

8. as each described URP among the claim 1-3, it is characterized in that the aminoacid that is selected from any type of glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) or proline (P) accounts among the described URP all amino acid whose about more than 30% separately.

9. as each described URP among the claim 1-3, it is characterized in that described URP comprises about contiguous amino acid more than 200.

10. as each described URP among the claim 1-3, it is characterized in that described URP contains 3,4,5 or 6 kind of dissimilar aminoacid.

11., it is characterized in that described URP contains 4,5 or 6 kind of dissimilar aminoacid as each described URP among the claim 1-3.

12. protein that contains just like each described one or more URP among the claim 1-3, wherein said one or more URP and described protein allos, described protein comprises one or more functional modules, and described functional module is selected from effect module, binding modules, N-terminal module, C-terminal module or its combination in any.

13. protein as claimed in claim 12, it is characterized in that described effect module is selected from cytokine, somatomedin, enzyme,-receptor, microprotein, hormone, erythropoietin, adenosine takes off the imines enzyme, asparaginase, arginase, interferon, growth hormone, growth hormone releasing hormone, G-CSF, GM-CSM, insulin, hirudin, the TNF-receptor, uricase, rasburicase, Axokine, the RNA enzyme, the DNA enzyme, phosphatase, Pseudomonas exotoxin, ricin, gelonin, the general enzyme of ammonia, the La Luoni enzyme, thrombin, thrombin, VEGF, general Lip river tropine, growth hormone, alteplase, interleukin, the IIV factor, the VIII factor, the X factor, the IX factor, streptodornase, glucocerebrosidase, follitropin, glucagon, thyrotropin, Nesiritide, alteplase, parathyroid hormone, Ah add'sing carbohydrase, La Luoni enzyme and methioninase.

14. a recombination of polynucleotide, it comprises the coded sequence of each described URP among the coding claim 1-3.

15. a recombination of polynucleotide, it comprises coding claim 12 or 13 described proteinic coded sequences.

16. a host cell, it comprises claim 14 or 15 described recombination of polynucleotide.

17. a carrier, it comprises claim 14 or 15 described recombination of polynucleotide.

18. a generation contains the method for protein of unstructured recombinant polymers (URP), described method comprises:

(i) provide the host cell of the recombination of polynucleotide that contains code for said proteins, described protein contains one or more URP, described URP substantially can not with the serum albumin non-specific binding, and described URP has following feature:

(a) contain at least 100 contiguous amino acids;

(b) contained glycine (G), aspartic acid (D), alanine (A), serine (S), threonine (T), glutamic acid (E) and proline (P) residue sum accounts among the described URP all amino acid whose about more than 80% among the described URP;

(c) at least 50% aminoacid of measuring described URP through the Chou-Fasman algorithm does not form secondary structure; And

(d) scoring of the T epi-position of described URP is less than-4;

(ii) under appropraite condition, cultivate described host cell in the suitable culture medium, so that express described protein by described polynucleotide.

19. method as claimed in claim 18 is characterized in that, described URP comprises glycine.

20. method as claimed in claim 18 is characterized in that, described URP comprises glutamic acid.

21., it is characterized in that described host cell is an eukaryotic cell as each described method among the claim 18-20.

22., it is characterized in that described host cell is a Chinese hamster ovary celI as each described method among the claim 18-20.

23., it is characterized in that described host cell is a prokaryotic cell as each described method among the claim 18-20.

24., it is characterized in that described URP comprises the alpha-non-natural amino acid sequence as each described method among the claim 18-20.

25., it is characterized in that described URP comprises and surpasses 200 contiguous amino acids as each described method among the claim 18-20.

26., it is characterized in that described URP comprises repetitive sequence as each described method among the claim 18-20.

27., it is characterized in that described protein is therapeutic protein as each described method among the claim 18-20.

28., it is characterized in that described protein comprises one or more modules that are selected from down group: binding modules, effect module, multimerization module, C-terminal module and N-terminal module as each described method among the claim 18-20.

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