WO2004001041A1 - 発現ベクター、宿主、融合タンパク質、融合タンパク質の製造方法及びタンパク質の製造方法 - Google Patents
発現ベクター、宿主、融合タンパク質、融合タンパク質の製造方法及びタンパク質の製造方法 Download PDFInfo
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- WO2004001041A1 WO2004001041A1 PCT/JP2003/008020 JP0308020W WO2004001041A1 WO 2004001041 A1 WO2004001041 A1 WO 2004001041A1 JP 0308020 W JP0308020 W JP 0308020W WO 2004001041 A1 WO2004001041 A1 WO 2004001041A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
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- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Definitions
- Expression vector host, fusion protein, method for producing fusion protein, and method for producing protein
- the present invention is directed to production of an expression vector, a host, a fusion protein, a protein, and a fusion protein that can prevent the recombinant protein from being expressed as an abnormal form such as an inclusion body and can be produced as a natural form in a soluble fraction.
- the present invention relates to a method and a method for producing a protein. Background art
- the properties of the recombinant protein obtained by integrating the relevant gene into a vector gene, transforming it into a host such as a bacterium, an yeast, or an insect cell, and expressing the protein are determined.
- the method of examining is common.
- the target protein When the target protein is expressed as an inclusion body that is an abnormal protein, the usual method for obtaining the normal form is to convert it to the normal form in vitro. That is, the inclusion body is collected from the host, solubilized with high-concentration guadin hydrochloride, urea, etc., and then diluted about 30 to 100 times with an appropriate buffer, etc., to refold the solubilized target protein. It is a way to make it. For example, antibodies are expected to be used in the medical field, etc., but if the recombinants are expressed in the cytoplasm of E. coli as a host, most of them will be expressed as insoluble inclusion bodies. It is known (Pluckthun, Biotechnology, 9, 545—, 1991).
- membrane proteins also have a high water-hydrophobic amino acid content due to the nature of being buried on the surface or inside of biological membranes, and often appear as inclusion bodies when expressed as recombinant proteins in the absence of membranes. It is known to be expressed. In many cases, expression does not even lead to expression of toxicity to cells.
- eukaryotic cells such as yeast or animal cells and to express it in the membrane fraction. Since this requires cost and labor, a simpler expression method is desired.
- the present invention can prevent the formation of an inactive abnormal protein at the time of recombinant protein production, and can produce a target protein in a natural and soluble form in large quantities and efficiently. It is an object of the present invention to provide an expression vector, a host, a fusion protein, a protein, a method for producing a fusion protein, and a method for producing a protein.
- the present invention provides: (a) a first coding region encoding a polypeptide having molecular chaperone activity; and (b) at least one restriction enzyme site into which a second coding region encoding a protein can be inserted.
- a first coding region encoding a polypeptide having molecular chaperone activity
- at least one restriction enzyme site into which a second coding region encoding a protein can be inserted.
- the first coding region is operatively linked to a promoter, and the restriction enzyme site is in the same reading frame as the first coding region, and Downstream of the region, or the restriction enzyme site is arranged such that the inserted second coding region is effectively linked to a promoter.
- the first code region is in the same decoding frame as the second code region and downstream of the second code region.
- the expression vector of the present invention is located between the first coding region and the region having at least one restriction enzyme site into which the second coding region can be inserted, and is translated in the same reading frame to obtain a protease digestion site. It is preferable to have a region.
- the expression vector of the present invention preferably incorporates a second coding region encoding a protein.
- the polypeptide having a molecular opening activity is preferably PPIase having a molecular opening activity.
- FKB P-type PPIase examples include archaeal-derived FKBP-type PPIase, trigger fatter type PPIase, FkpA type PPIase, and FKBP52 type PPIase.
- cycloburine-type PPIase examples include CyP40 type PPIase.
- the Purbrin-type PPIase includes a SurA type PPIase.
- Examples of the PPIase having the molecular chaperone activity include an IF domain of an archaebacterium-derived FKB P-type PPIase and / or a PPIase containing a C-terminal domain, and an N-terminal domain of a trigger factor type PPase.
- the second coding region may be a force having a nucleotide sequence encoding a monoclonal antibody or a nucleotide sequence encoding a membrane protein. It is preferred to have.
- a host containing the expression vector of the present invention is also one of the present invention.
- the host of the present invention is preferably E. coli.
- a fusion protein containing the polypeptide having the molecular chaperone activity and the protein encoded by the second coding region is also one of the present invention.
- the fusion proteins of the present invention preferably contain a protease digestion site.
- the method for producing the fusion protein of the present invention is also one of the present invention.
- a host containing the expression vector of the present invention is cultured under the expression conditions of the expression vector, and the fusion protein is expressed in the cell, or the expression vector of the present invention is used.
- a region that is transcribed and translated into a signal sequence at the 5th end of the first coding region or the 55th end of the second coding region to form a signal sequence, and the host containing the obtained expression vector is subjected to expression conditions for the expression vector.
- the above fusion protein is cultured in a cell-free translation system by expressing the fusion protein in a periplasm or a culture medium or expressing it in a cell-free translation system.
- the carrier is recovered after adsorbing the fusion protein to a carrier supporting macrolide, cyclosporin, juglone, or a compound thereof that inhibits PPIase activity. Is preferred.
- FIG. 1 is a diagram showing the gene arrangement of a vector TcFK fusion 2 for producing a fusion protein with a short type FKBP-type PPIase derived from Thermococcus sp. KS-1.
- FIG. 2 shows the expression of TcFKBP18 when TcFK fusion 2 was used.
- FIG. 3 is a diagram showing an electrophoresis pattern of a host-derived protein.
- FIG. 4 is a diagram showing the expression of a fusion protein of a mouse-derived anti-nitrilysozyme (HEL) Fab antibody fragment and TcFKBP18.
- FIG. 5 is a diagram showing expression of a mouse-derived anti-nitrilysozyme (HEL) Fab antibody fragment alone.
- FIG. 6 is a diagram showing mouse-derived anti-ditrilysozyme (HEL) scFv fragment and its expression of a fusion protein with TcFKBP18.
- FIG. 7 is a diagram showing a fusion protein of a purified mouse-derived anti-HEL scFv and TcFKBP18 and the result of thrombin treatment.
- FIG. 8 is a diagram showing the activity of mouse-derived anti-HEL scFv obtained as a result of expression by the ELISA method.
- “effectively linked to a promoter” means that the first coding region is linked to a promoter so that a polypeptide having a molecular shuttling activity is normally transcribed.
- PPIase having molecular chaperone activity includes those having substantially equivalent functions. That is, it includes substantially equivalent polypeptides, polypeptides containing at least a part thereof, and those obtained by modifying some amino acids to other amino acids.
- the “domain” includes a domain having substantially the same function.
- the expression vector of the present invention contains (a) a first coding region encoding a polypeptide having molecular chaperone activity.
- the above molecular chaperone activity means that the denatured protein is
- the activity refers to the activity of denaturing or inhibiting the irreversible aggregation of denatured proteins.
- rhodanese, quenate synthase, malate dehydrogenase, glucose-16-phosphate dehydrogenase, etc. are used as model enzymes (Kawata, Bioscience and Industry-1 56, 593—, 1998).
- a protein denaturant such as 6 M guanidine hydrochloride
- the denaturant is diluted with a buffer containing the substance to be assayed. It is possible to evaluate the molecular opening activity of the object.
- the polypeptide having the molecular chaperone activity is not particularly limited, and examples thereof include PPI ases having a molecular chaperone activity such as archaeal-derived FKB P-type PPIase; Sumonoreheat shock protein, Prephonoredin, DnaK :, DnaJ, GrpE, HSP90 and the like.
- the small heat shock protein is, Sabuyuni Tsu city of about 1 5 ⁇ 30 kD a can, 2: gathered to 32 about to take a giant molecular structure, have been reported to have a chaperone activity (J akob, J. Biol. Chem 268, 15 17-, 1999). Crystallin having a region with high homology to the C-terminal region also has the same properties as the small heat shock protein, and any of them can be applied to the expression vector of the present invention.
- the above-mentioned shark mouth nin has a characteristic structure consisting of a total of 14 to 18 subunits, in which a donut-shaped structure in which 7 to 9 subunits are connected in a ring form overlaps in two stages.
- the lip mouth captures the denatured protein in the recess of the donut structure and promotes protein refolding with consumption of nucleotides such as ATP.
- nucleotides such as ATP.
- eubacteria-derived group 1 shadinin it promotes the protein folding reaction with the binding of GroES (heat shock protein 10) as a cofactor.
- the above-mentioned prefoldin is a molecular chaperone found as a factor involved in the protein folding of eukaryotic tupurin (L opez, J. Struct. Biol. 135, 219—, 2001). It is known that it forms a dimer and has in vivo activity that interacts with denatured protein (Siegert, Cell 1103, 621-, 2000) .
- the above-described homologues of DnaK, DnaJ, and GrE are molecular haploids that are widely present regardless of the species of organism and are considered to be involved in protein folding.
- HSP90 heat shock protein 90
- chaperone-like activity Rasey, J. Biol. Chem. 275, 17857-, 2000.
- the homologue, a part thereof, or a polypeptide containing them can be used.
- PPI ase having the molecular Shapeguchin activity is one of the protein folding factors involved in Fonorete Ingu the end tooth 0 click quality, Fall di ring intracellularly Of the amino acids in the developing target protein, the N-terminal side of the proline residue It has the activity of catalyzing the cis-trans isomerization reaction of peptide bonds (PPase activity).
- PPIase having a molecular chaperone activity is particularly preferable.
- FK506BindingProtein type FKBP type
- cyclophilin type a PPIase whose activity is inhibited by FK506, one of the immunological inhibitors, and homologs thereof.
- the cyclophilin-type PPIase is a PPIase or a homologue thereof that is sensitive to cyclosporine, another immunosuppressive agent.
- parbulin-type PPIase is not sensitive to any immunosuppressive agent, and is a PPIase or a homolog thereof whose activity is inhibited by juglone (jugone). These three types of PPI ase have almost no homology in the amino acid primary sequence.
- the PPI ase having the molecular chaperone activity may be any of the above three types of PPI ase.
- FKB P-type PPIase examples include archaebacterial FKB P-type PPIase, trigger factor type PPIase (Hang, Protein Sci. 9, 1254—, 2000), FkpA type PPI Microbiol. 39, 199-2001, FKB P52 type PPI ase (Bose, Science 274, 171 15—, 196) No.
- cyclophyrin type PPIase examples include CyP40 type PPIase (Pirkl, J. Mo 1. Biol. 308, 795—, 2001) and the like.
- Purblin type PPIase examples include SurA type PPIase (Behrens, EMBO J. 20, 285—, 2001).
- the molecular chain activity is the activity originally found in the protein chain of the Shainin and the Dna / DnaJ / GrE system, which are originally known as one of the molecular chains. They function to support the biosynthetic polypeptides in cells to fold correctly. At that time, hydrolysis of high energy substances such as ATP is required.
- the archaeal-derived FKBP-type PPIase is excellent in that it does not require a hydrolysis reaction of the high-energy substance when exerting its molecular chaperone activity.
- the archaebacteria-derived FKB P-type PPIase can be broadly classified into two types depending on the molecular weight. One is a short type having a molecular weight of about 16 to 18 kDa, and the other is a long type having a molecular weight of about 26 to 33 kDa.
- the archaeal-derived FKBP-type PPIase used in the present invention may be either a short type or a long type: PKBKB type PPIase.
- the short type tends to have stronger molecular chaperone activity, and the expression of the recombinant protein tends to decrease as the molecular weight of the protein increases.
- a short type archaebacterium-derived FKB P-type PPIase is preferred.
- the range of the molecular weight described above is the molecular weight range of PPI ase that has been found so far, and the archaeal-derived FK? Type? I ase is not limited to this molecular weight range, and may be any one that substantially belongs to the same group.
- the archaeal-derived FKB P-type PPIase is not particularly limited, and may be of any archaeal origin.
- Is a Methanococcusthermolit hotr an archaebacterium derived from thermophilic and hyperthermophilic bacteria S-1 derived from ophicus, from Thermo coccussp.
- S-1 derived from Me thanococcus jannaschii, derived from Methanosarcina ma zei which is a normal temperature deciduous archae, derived from Me thanosarcinaacetivora ns, derived from Me thanosarcinabarkeri, etc.
- the long type has been found in the genomes of most archaebacteria as a result of genome analysis and other analyses.
- the long type is derived from Pyrococcushorikoshii, a thermophilic and hyperthermophilic archaeon, and Aeropyr um per ⁇ ⁇ x, aulfolooussolfataricus, Me thanococcus jannaschii, Archaeoglobusfulgidus, Me thanobacteri um autotrophic um Th e rmo 1 a sma acidophi 1 um, Halobacterium cutirubrum which is a halophilic bacterium, etc.
- the archaeal FKB P-type PPI ase has an FKB P domain and an IF domain involved in the binding between PPIase activity and FK506, and is a long-type archaeal FKB P-type PPI ase.
- it further has a C-terminal domain (Ma ruyama and Furutani, Front Biosci. 1, D 821—, 2000).
- the above-mentioned IF domain (Insertintheflap; Suzuki, J. Mo. Biol. 328, 1149—, 2003) contains the amino acid sequence inserted into the amino acid sequence constituting the FKBP domain on the amino acid primary sequence.
- the FKB P-type PPIase is associated with the FK506-binding domain of the FKB P domain and the IF domain in the molecular shut-in activity of the short-type archebacterium FKB P-type PPIase (Fu rutani, Bioch em istry 39, 28 22—, 2000; I deno, Bioch em J 357, 465—, 2001).
- the C-terminal domain of the long-type archebacterium FKB P-type PPIase is involved in the molecular shut-in activity together with the two domains (I DENO, Eur JBiooch). em. 26 7, 31 39—, 2000).
- an archaeal-derived FKBP-type PPIase has an IF domain, and a PP containing a Z or C-terminal domain.
- PPI a se If it is 1 a se, it can be used as PPI a se having the above-mentioned molecular opening activity.
- a chimeric PPIase in which an IF domain or C-terminal domain is introduced into human FKBP12, which is a PPIase that originally has no molecular chaperone activity, by protein engineering should be used as the PPIase having the above molecular chaperone activity.
- the region from proline 78 to tyrosine 146 in SEQ ID NO: 1 and the region from proline 78 to 141 glutamic acid in SEQ ID NO: 2 Each region corresponds to the IF domain (I DENO, Eur JBiochem. 267, 3139—, 2000).
- the region from 157 isoloisin to the C-terminal corresponds to the C-terminal domain (I DENO, Eur JBiooch em. 26 7, 3 1 39-, 2000).
- the homology of each domain can be determined by using multiple alignment software such as Clusta1W.
- the trigger-factor PPIase is a PPIase found on the genome of almost all bacteria.
- the trigger factor type PPIase is not particularly limited, and includes, for example, Escherichia coli, Myco ⁇ 1 as ma genitalium, Bacillussubtilis, Salmonella enterica, and Taphy1ococcus. aureus, Mycobacteri um lerae, A grobacteri um tum efacium, L actococcus 1 actis, Camp yrobacter jejuni, Streptococcus pyogenes, and Corynebacterium diphthe riae.
- the trigger factor type PPIase used in the present invention may be any of the trigger factor type PPIases as long as they belong to a group recognized to be substantially the same as the bacterial trigger factor in the amino acid sequence. Is also good.
- the amino acid sequence of Escherichia coli-derived trigger fat type PPIase is shown in SEQ ID NO: 3, and the nucleotide sequence is shown in SEQ ID NO: 4.
- the trigger factor type PPlase has an FKB P domain involved in binding between PPIase activity and FK506 as an intermediate domain, and has two domains on its N-terminal side and C-terminal side, respectively ( Z arnt, J. Mo 1. Biol. 27 1, 8 27-, 1997). It is known that the molecular triggering activity of the trigger factor type PPIase is independent of the PPIase activity, similar to the archaeal FKB P-type PPlase, and its N-terminal domain and C-terminal It has been suggested that it is an effect of either one of the domains or both.
- any PPIase containing the N-terminal domain of the trigger factor type PPlase and / or a Cterminal domain can be used as a PPIase having the above molecular chaperone activity.
- the N-terminal domain and C-terminal domain of the trigger one-factor type PPIase in SEQ ID NO: 3, the region from methionine 1 to arginine 144 corresponds to the N-terminal domain, The region from the vicinity of luranin to the C-terminal corresponds to the C-terminal domain (Z arnt, J. Mo 1. Biol. 271, 827—, 1997).
- the homology of each domain can be determined by using multiple alignment software such as C1usta1W.
- Fkp A-type PPIase and Sur A-type PPIase are expressed in the periplasmic region of Gram-negative bacteria such as E. coli. PI as ⁇ .
- the Fkp A-type PPIase is an FKB P-type PPIase whose activity is inhibited by FK506, whereas the Sur A-type PPIase is FK506 and another immunosuppressant cyclosporin It is one of the parbulin-type PPIase homologs that is insensitive to any of the immunosuppressive agents.
- These two PPIases are also known as proteins that exhibit molecular shuttling activity (Ramm, J. Biol. Chem.
- the FkpA-type PPIase and the SurA-type PPIase have been found not only in the genome of Gram-negative bacteria but also in homologs of eukaryotes such as yeast.
- F k pA type used in the present invention Type of PPI ase and S11? ? 1 & se is not particularly limited. Hemo philusinfluenzae, Ralstoniasolanacearum and the like. In addition, not only those derived from bacteria but also PPI ases derived from any organism may be used as long as they belong to the same group and have substantially the same function.
- the FkpA type PPIase the amino acid sequence of Escherichia coli is shown in SEQ ID NO: 5, and the nucleotide sequence is shown in SEQ ID NO: 6.
- the amino acid sequence of Escherichia coli is shown in SEQ ID NO: 7
- the nucleotide sequence is shown in SEQ ID NO: 8.
- the FkpA-type PPIase has a FKBP domain on the C-terminal side and an N-terminal domain other than the FKBP domain (Arie, MoI. Microbiol. 39, 199-, 200). 1 year). It is known that the molecular chaperone activity of the FkpA-type PPIase is also independent of the PPIase activity, suggesting the involvement of its N-terminal domain. In the present invention,? Type? If the PPI ase contains the N-terminal domain of Iase, It can be used as a PPIase having peron activity. As the N-terminal domain of the FkpA-type PPIase, in SEQ ID NO: 5, the region near aspartic acid No. 120 from the N-terminal corresponds to the N-terminal domain (Arie, Mo 1. Microbiol. 39). , 1 991-1, 200 1).
- the SurA-type PPIase also has a domain with high homology between the parbulin-type PPIase and the other N-terminal domain on its C-terminal side. It has been suggested that the N-terminal domain is involved in the molecular shut-down activity of the Sur A-type PPIase in addition to the domain with high homology between the purpurin-type PPIases (Behrenns, EMBO J. 20, 285—, 2001). In the present invention, any PPIase containing the N-terminal domain of the above-mentioned SurA type PPIase can be used as the above-mentioned PPIase having molecular chaperone activity.
- the region near the 175th asparagine from the N-terminal corresponds to the N-terminal domain.
- the homology of the N-terminal domain can be determined by using multiple alignment software such as C1usta1W.
- the FKBP52 type PPIase and CyP40 type PPIase are both PPIases found in eukaryotes.
- the FKBP 52 type PPIase is an FKBP type PPIase of about 52 kDa, and is also called p59 or HSP56. Its amino acid sequence has a configuration in which two regions having high homology to human-derived 12 kDa FKB P-type PPIase are linked in tandem, and a region containing a calmodulin-binding site is linked to the C-terminal side (R atajczat J. Biol. Chem. 268, 1387—, 1993).
- the CyP40-type PPIase has a molecular weight of about 40 kDa, and is one of cyclosporine-type PPIases that are sensitive to cyclosporine, an immunosuppressant. Each of them is characterized by having a domain responsible for PPIase activity at its N-terminus and a domain containing a tetratricopeptide repeat (TPR) that binds to one of the heat shock proteins, HSP90, at its C-terminus.
- TPR tetratricopeptide repeat
- the above-mentioned FKBP52 type PPIase and CyP40 type PPIase are not particularly limited, and include, for example, those derived from eukaryotes such as human, mouse, red pepper, rabbit, and rat. Further, the FKBP52 type PPIase and CyP40 type PPIase used in the present invention are not only those derived from eukaryotes but also belong to a group recognized as PPIases having substantially the same functions. It can be a PPIase from any organism as long as it belongs to it.
- an amino acid sequence derived from human is shown in SEQ ID NO: 9, and its base sequence is shown in SEQ ID NO: 10.
- the amino acid sequence of the human-derived PPIase is shown in SEQ ID NO: 11, and the base sequence is shown in SEQ ID NO: 12.
- the molecular terminal activity of FKBP52 type PPIase and CyP40 type PPIase is related to the respective C-terminal domains including TPR ⁇ .
- any PPIase containing the C-terminal domain of FKB P52 type PPIase and CyP40 type PPIase can be used as a PPIase having the above molecular chaperone activity.
- the above C-terminal domain is a region from near glutamic acid 264 to the C-terminus in SEQ ID NO: 9 in human FKBP52 type 2 PPIase, of which the region from glutamic acid 264 to near isoloisin 400 is particularly important. is there.
- human CyP40 type PPIase the region from the vicinity of leucine 184 to the C-terminal corresponds to the C-terminal domain.
- the PPIase having molecular chaperone activity used in the present invention can be suitably used as long as it is a PPIase having equivalent molecular chaperone activity, other than those exemplified above.
- Examples include a pta-derived 18 kDa cyclophilin-type PPIase (Ou, Protein Sci. 10, 2346—, 2001) whose molecular chaperone activity has recently been re-evaluated.
- the expression vector of the present invention contains (b) a region having at least one restriction enzyme site capable of inserting a second coding region encoding a protein. It is.
- the second coding region is a region having a base sequence encoding a target protein to be expressed using the expression vector of the present invention.
- the second coding region used in the present invention is not particularly limited, and examples thereof include those having a nucleotide sequence encoding an antibody such as a monoclonal antibody and those having a nucleotide sequence encoding a membrane protein.
- the above antibody may be an antibody derived from any animal species, and two or more fragments thereof such as full length antibody, a fragment thereof, Fab, Singlechain Fv (scFv), etc. are linked by a linker peptide. Polypeptides and the like are also included in the above antibodies.
- the above antibodies may be of any subclass.
- Antibodies are macromolecules with a molecular weight exceeding 100,000, and are widely used as analytical reagents, in vitro diagnostics, etc., utilizing their ability to specifically bind to specific antigenic substances. High utility value.
- the portion that contributes to the binding between the antibody molecule and the antigenic substance is called the V region (variable region), and is composed of the V region of the heavy chain and the V region of the light chain.
- Methods for obtaining an antibody against a specific antigen include a method of immunizing a laboratory animal such as a rat or a heron with an antigenic substance to obtain an antibody (polyclonal antibody) contained in the serum, and a method of obtaining a monoclonal antibody described below. Is common.
- Monoclonal antibodies are antibodies produced by a single clone of antibody-producing cells, characterized by a uniform primary structure. Monoclonal antibodies have become easier to produce with the establishment of the hybridoma technology by Koehler and Milstein.
- a predetermined antigenic substance is administered to an experimental animal such as a mouse to perform immunization immunization.
- spleen cells that have acquired the ability to produce the antibody against the antigen are taken out and fused with appropriate tumor cells such as myeloma to produce a hybridoma.
- a hybridoma producing the desired antibody is selected by screening using an appropriate immunoassay such as ELISA.
- a hybridoma producing the desired monoclonal antibody is established by cloning using a limiting dilution method or the like. After culturing the thus established hybridoma in a suitable medium, the medium containing the metabolites is separated by chromatography or the like, whereby the desired monoclonal antibody is obtained. can get.
- these methods inevitably require the intervention of experimental animals because they utilize the in vivo biological reaction of immunization of animals. Therefore, laboratory animals must be bred and maintained, which requires enormous labor and large costs. In addition, this method cannot always produce monoclonal antibodies against all antigenic substances, and involves trial and error.
- the membrane protein is not particularly limited, and includes, for example, a receptor for a physiologically active substance.
- Receptors for the above-mentioned physiologically active substances selectively respond to various extracellular substances and transmit a variety of signals into cells, so elucidating their functions is very likely to lead directly to drug discovery. Attention has been paid.
- These membrane receptor proteins form a structurally well-conserved family, which can be broadly classified into three types: ion channel intrinsic type, tyrosine kinase type, and G protein-coupled type. .
- ion channel endogenous type when a ligand binds to a receptor, an ion channel existing in the receptor itself is opened, and Na + and Ca 2 + are moved into the cell using an ion gradient inside and outside the cell.
- Type. The tyrosine kinase form amplifies the signal by converting ligand binding into increased phosphorylation activity, triggering a series of cascades.
- the G protein-coupled type is a receptor It has no ion channels or enzymatic activity, and transmits information from ligand binding into cells via G proteins. A number of drugs have been developed that target membrane receptor proteins, but many target G protein-coupled receptors (GPCRs).
- a vector having a base sequence encoding a membrane protein is incorporated as the second coding region into the expression vector of the present invention, a large amount of recombinant protein can be prepared at low cost.
- the restriction enzyme site is also called a multiple cloning site.
- the region having the above restriction enzyme site is a region into which a gene encoding a target protein is inserted as a second coding region.
- the first coding region is effectively linked to a promoter, and the restriction enzyme site is in the same reading frame as the first coding region;
- the second coding region in which the restriction enzyme site has been inserted is arranged so as to be effectively linked to a promoter, and the first coding region is located in the first coding region. It is in the same decoding frame as the second code area, and is downstream of the second code area.
- the above promoter is not particularly limited, and examples thereof include a P1ac promoter, a Ptac promoter, an Xy1A promoter, an ArabB promoter, a lambda promoter, a T7 promoter, a gall / gallO promoter, and an nmt1 promoter. And a polyhedrin promoter, a mouse meta-mouth motor and the like.
- a second coding region encoding the target protein is inserted into the above restriction enzyme site, and the expression vector in which the second coding region is incorporated is inserted.
- the first coding region is effectively linked to a promoter, and the restriction enzyme site has the same reading frame as the first coding region.
- the restriction enzyme site has the same reading frame as the first coding region.
- the two coding regions are arranged so as to be effectively linked to a promoter, and the first coding region is in the same reading frame as the second coding region and is downstream of the second coding region.
- the target protein encoded by the second coding region is a molecule chaperone activity It is expressed as a fusion protein with Poribe peptide having.
- the expression vector of the present invention is located between the first coding region and a region having at least one restriction enzyme site into which the second coding region can be inserted, and is translated in the same reading frame. May have a region that becomes a protease digestion site.
- the protease digestion site comprises a polypeptide having a molecular chaperone activity encoded by the first coding region and a second coding region, which are obtained by expression of an expression vector in which the second coding region is incorporated into the expression vector of the present invention.
- the fusion protein serves as a peptide linker that connects the polypeptide and the protein.
- the obtained fusion protein has a protease digestion site, the fusion protein is easily digested by the action of a protease, and the polypeptide having a molecular chaperone activity encoded by the first coding region is obtained.
- the target protein encoded by the second coding region can be obtained by separating the protein encoded by the second coding region from the protein encoded by the second coding region.
- the protease is not particularly limited, and includes, for example, thrombin, factor Xa, precision protease and the like. These proteases are commercially available from Pharmacia Biotech. It is also possible to cut off the target protein using the intin's self-protein splicing function.
- the length of the base sequence encoding the protease digestion site is not particularly limited, and is preferably about I15 to 90 bases. It is preferable to include a large number of base sequences that become neutral amino acids.
- the expression vector of the present invention may contain other known nucleotide sequences.
- the other known base sequences are not particularly limited, and include, for example, a stability leader sequence for imparting stability of an expression product, a signal sequence for imparting secretion of an expression product, a neomycin resistance gene, a kanamycin resistance gene, and chloramphene. Marking sequences capable of conferring phenotypic selection in transformed hosts, such as a call resistance gene, an ampicillin resistance gene, and a hygromycin resistance gene.
- the expression vector of the present invention may be designed in such a form that the obtained fusion protein is bound to an immobilized carrier via an appropriate ligand. Thus, after expression, its purification can be simplified.
- FKB506-type PPIase when used as a polypeptide having molecular chaperone activity, FKB506-type PPIase can be used for FK506-rapamycin, a carrier supporting its analogous compound can be used, and cyclophilin-type PPIase can be used for cyclosporine.
- the purification of the fusion protein can be simplified by using a carrier carrying the analogous compound and Jug 1 one or a carrier carrying the analogous compound in the case of perbulin-type PPIase. .
- the expression vector of the present invention was designed to have a tag of about 6 histidine residues on the N-terminal side of the polypeptide having the molecular chaperone activity, the obtained fusion protein chelated metals such as nickel. Since it binds to the carrier via a histidine residue, the use of the carrier makes it possible to easily separate the host-derived protein and the fusion protein. Further, by causing a protease to act on the fusion protein bound to the carrier, the protease digestion site is digested, and only the target protein can be easily released from the carrier.
- the fusion protein can be released from the carrier without the action of protease.
- a method using creatathion-1s-transferase or a part thereof as a tag and purification by affinity chromatography using a glutathione resin, or a method using a maltose-binding protein or a part thereof as a tag and a maltose resin A purification method or the like can also be used.
- affinity with antibodies can be used You.
- the above various tags may be designed on either the N-terminal side or the C-terminal side of the fusion protein, or may be designed on both sides. As these gene manipulation methods and affinity purification methods, methods known to those skilled in the art can be used.
- the polypeptide having the molecular sharing activity encoded by the first coding region and the second A fusion protein with the protein encoded in the coding region is obtained.
- a fusion protein containing the polypeptide having the molecular chaperone activity and the protein encoded by the second coding region is also one of the present invention.
- the target protein can be easily cut out from the fusion protein by digesting the obtained fusion protein with a protease.
- the fusion protein of the present invention preferably contains a protease digestion site.
- the expression vector of the present invention is introduced into a host and used for expression of the target protein.
- a host containing the expression vector of the present invention is also one of the present invention.
- the host is not particularly limited, and includes, for example, prokaryotes such as bacteria, yeast, fungi, plants, insect cells, mammalian cells, and the like.
- the characteristics of the host used and the expression vector must be compatible.
- the expression vector may be a promoter isolated from the genome of a mammalian cell, such as the mouse metacholinechlein motor, a baculovirus promoter, a vaccinia virus 7.5K promoter, or the like. It is preferred to use a promoter isolated from a virus that grows in mammalian cells.
- prokaryotic organisms such as Escherichia coli are preferable.
- the method for introducing the expression vector of the present invention into a host is not particularly limited, and various known methods can be used. For example, phosphate transfection, electroporation, ribosome fusion, and nuclear injection as transfusions , Virus or phage infection.
- a large amount of fusion protein can be obtained.
- Such a method for producing the fusion protein of the present invention is also one of the present invention.
- a host containing the expression vector of the present invention is cultured under the expression conditions of the current vector, and the fusion protein is expressed in the cell, or the expression vector of the present invention is used.
- a region which is transcribed and translated into a signal sequence at the 5 'end of the first coding region or at the 5th end of the second coding region to become a signal sequence It is preferable that the above fusion protein is cultured in a culture medium and expressed in the periplasm or medium, or the expression vector of the present invention is expressed in a cell-free translation system.
- the expression of the fusion protein may be cytoplasmic, periplasmic or in a culture medium.
- the fusion protein can be transformed into a periplasm or a periplasm. It can be secreted and expressed in the medium.
- expression of the polypeptide encoded in the first coding region can be improved by using a polypeptide originally present in a membrane in a cell.
- polypeptides originally present on the membrane in the cell include the FKBP type PPIase, FkpA type PPIase, and the purin type PPIase, SurA type PPIase, and the like. These PPI ases are naturally present in the periplasm of Gram-negative bacteria and are proteins involved in protein folding.
- the protein encoded by the second coding region is a membrane protein, an antibody, or the like, since these proteins are originally expressed outside the cytoplasm, the FkPA-type PPIase and the SurA-type PPI Expression is improved if expressed by fusing with ase or the like.
- a cell-free translation system (Spirin, AS, 1 99 1, S cience 1 1, 2656-2664: F alcone, D. eta 1., 1991, Mo 1. Cell. Biol. 11, 1, 2656-26664)
- the protein is expressed as a soluble protein.
- the carrier may be recovered after adsorbing the fusion protein to a carrier supporting PPIase activity-inhibiting macrolide, cyclosporin, juglone, or a compound similar thereto. preferable.
- the binding between the PPIase having the molecular chaperone activity and the inhibitor is strong, and the expressed fusion protein can be purified using this affinity. For example, if beads using macrolide such as FK506 on agarose gel carrier are used,? 1 ⁇ 8 bun type? ? A fusion protein with Iase can be specifically bound.
- the target protein can be obtained by digesting the fusion protein obtained by the method for producing a fusion protein of the present invention with a protease that digests a protease digestion site.
- a method for producing the protein encoded by the second coding region is also one of the present invention.
- a target protein is expressed as a fusion protein by linking the target protein together with a polypeptide having molecular chaperone activity with a peptide linker, and thereby, a poorly expressed protein which is originally expressed as an abnormal type Can be expressed in large quantities as a natural soluble form, and its productivity can be greatly increased.
- the target protein is an antibody
- Example 1 Construction of an expression vector for fusion with a short-type FKB P-type PPIase (TcFKBP18) derived from the hyperthermophilic archaeon T thermococcus p. KS-1 Expression of T c FKB P 18 (I deno, Biochem. J. 357, 465_, 2001) having an active plasmid p EFE l_3 (I ida, Gene 222, 249—, 1998) The ⁇ ⁇ type was obtained, and the TcFKBP18 gene fragment was amplified by PCR.
- TcFKBP18 short-type FKB P-type PPIase
- TcFu_F1 and TcFU-R2 shown in Table 1 restriction enzyme sites were provided at both ends of the amplification product.
- Throm-F2 and its complementary strand were designed as a nucleotide sequence encoding a linker for cleaving the TcFKBP18 fusion protein into TcFKBP18 and the target protein by protease. did.
- ThromF2 has a SpeI site on its 5 'side and an EcoRI site on its 3' side ( Figure 1).
- Ding 11 1 "0111-2 has a BamHI site and an NdeI site downstream of the DNA sequence in the thrombin-cleaved region, so that the gene fragment of the target protein can be replaced with these restriction enzymes. By introducing it using the site, a fusion protein with TcFKBP18 can be obtained (Fig. 1).
- TcFKBP18 gene fragment and the DNA fragment encoding the thrombin-cleaved portion were treated with the respective restriction enzymes, and were treated with NcoI / EcoRI beforehand.
- DNA manufactured by Novagen
- the obtained plasmid for expressing the TcFKBP18 fusion protein was designated as TcFK fusion 2.
- Expression vectors for fusion (Example 2) from Escherichia coli trigger factor-type PPI a s e (TF) Construction
- the TF gene excluding the stop codon was amplified by PCR from E. coli K12 strain.
- TF-F1 and TF-R1 shown in Table 1 primers for PCR, restriction enzyme sites were provided at both ends of the amplification product.
- the TcFK fusion 2 prepared in Example 1 was treated with NcoI / SpeI, and the vector from which the TcFKBP18 gene had been removed was agaro-treated. Purified by Suge electrophoresis.
- the ⁇ 7 blue ⁇ vector containing the TF gene was treated with ⁇ coI / SpeI, and the excised TF gene was recovered. Ligation of the obtained TF gene and the above vector. One vector containing the full length TF gene was recovered.
- a TF fusion protein expression system in which the TcFKBP18 gene in TcFKfusion 2 of Example 1 was replaced with the TF gene was constructed.
- the obtained plasmid for expressing a TF fusion protein was designated as TFf2.
- the FKBP52 gene excluding the stop codon from human cDNA library was converted to PCR. Amplified.
- FK52-F1 and FK52-R1 shown in Table 1 as PCR primers, restriction enzyme sites were provided at both ends of the amplification product.
- TcFKfusicon2 prepared in Example 1 was treated with NcoI / SpeI, and the vector excluding the TcFKBP18 gene was purified by agarose gel electrophoresis.
- the pT7 blue T vector containing the hFKBP52 gene was treated with NcoI / SpeI, and the excised hFKBP52 gene fragment was recovered.
- the obtained hFKBP52 gene fragment was ligated to the above vector, and a vector containing the full length hFKBP52 gene was recovered.
- a fusion protein expression system with hFKBP52 in which the TcFKBP18 gene in TcFKfusion2 of Example 1 was replaced with the hFKBP52 gene was constructed.
- the obtained plasmid for expressing the hFKBP52 fusion protein was designated as FK52f2.
- the pT7 blue T vector containing the hCyP40 gene was treated with NcoI / SpeI, and the excised hCyP40 gene was recovered.
- the obtained hCyP40 gene and the above vector were ligated, and a vector containing the full length hCyP40 gene was recovered.
- a fusion protein expression system with hCyP40 in which the TcFKBP18 gene in TcFK fusion 2 of Example 1 was replaced with the hCyP40 gene was constructed.
- the obtained plasmid for expressing the hCyP40 fusion protein was designated as CP40f2.
- the F kp A gene excluding the stop codon from E. coli CTF073 was Amplified.
- FKPA-F1 and FKPA-R1 shown in Table 1 primers for PCR, restriction enzyme sites were provided at both ends of the amplification product. After introducing the PCR product into the pT7 blue T vector, it was confirmed that the sequence was not different from the registered information.
- the TcFK fusion 2 prepared in Example 1 was treated with NcoI / SpeI, and the solid excluding the TcFKBP18 gene was purified by agarose gel electrophoresis.
- the pT7 pull-T vector containing the FkpA gene was treated with Nco1 / SpeI, and the excised FkpA gene was recovered.
- the obtained FkpA gene and the above vector were ligated, and a vector containing the full length FkpA gene was recovered.
- a fusion protein expression system with FkPA in which the TcFKBP18 gene in TcFKfusion 2 of Example 1 was replaced with the FkpA gene was constructed.
- the obtained plasmid for expressing the FkI) A fusion protein was designated as FkAf2.
- Example 6 Expression vector construction for fusion with Escherichia coli-derived Sur A type PPIase
- the pT7 pull-T vector containing the SurA gene was treated with NcoI / SpeI, and the cut-out SurA gene was recovered.
- the obtained SurA gene was ligated to the above vector, and a vector containing the entire SurA gene was recovered.
- a fusion protein expression system with SurA in which the TcFKB ⁇ 18 gene in TcFKfusicon2 of Example 1 was replaced with the SurA gene was constructed.
- the resulting SurA fusion protein expression plasmid was designated SurAf2.
- Example 7 Expression of TcFKB ⁇ 18 using TcFK fusion 2 E. coli BL 21 (DE 3) strain was transformed with Tc FK fusion 2 prepared in Example 1.
- 70 OmL of 2 X YT medium Yeast Extruct 16 g / L, BACTO TRYPTON 20 g / LNaC 15 g / L, Ampicillin 100 igZmL, pH 7.5
- Two to three loops of recombinant E. coli were inoculated. After rotating culture (110 rpm) at 35 ° C for 24 hours, the cells were collected by centrifugation (lOOO rpmX I Omin).
- the obtained cells were suspended in 2 OmL of 25 mM HEPES buffer (pH 6.8) containing ImM EDTA, and stored frozen at 120 ° C.
- the obtained bacterial solution was sonicated, centrifuged, and separated into a supernatant (soluble fraction) and a sediment (sediment fraction).
- the precipitate fraction was further purified to an inclusion body fraction by 4%.
- the membrane components are solubilized, and the inclusion body components precipitated by centrifugation are removed. Collected. This operation was repeated twice, and the obtained precipitate was used as an inclusion body fraction.
- Example 8 Expression of fusion protein consisting of TcFKBP18 and mouse-derived anti-nitrilysozyme (HEL) Fab antibody fragment (D1.3) Expression of mouse-derived anti-HE LF ab antibody fragment Plasmid p EHELF ab—1 (I deno, Appl. Env. Microbiol. 68, 464—, 2002) was treated with Ndel / B pull 02 I and electrophoresed on an agarose gel. Anti—HEL Fab antibody fragment gene fragment was purified. This DNA fragment was ligated to TcFK fusion 2, which had been previously treated with NdeI / Bpull02I.
- the heavy chain portion of the Fab is expressed as a fusion protein with TcFKBP18, and the light chain portion is expressed alone without becoming a fusion protein.
- the obtained plasmid was incorporated into Escherichia coli in the same manner as in Example 7 to obtain a transformant thereof. This bacterium was cultured and recovered in the same manner as in Example 7, and stored frozen at -20 ° C.
- the obtained bacterial solution was sonicated, centrifuged, separated into a supernatant (soluble fraction) and a precipitate (sediment fraction), and subjected to SDS-PAGE in the same manner as in Example 7.
- the SDS-PAGE gel was used to specifically detect the expressed Fab by staining with Coomassie brilliant pull (CBB) and Western blotting using an anti-D1.3 antibody derived from egret as the primary antibody. .
- CBB Coomassie brilliant pull
- No band corresponding to the fusion protein of Fab and TcFKBP18 was observed in the soluble fraction and the precipitated fraction of Escherichia coli, the host bacterium, neither by CBB staining nor by Western blotting. ( Figure 3).
- the expression plasmid pEHELFab-1 of a mouse-derived anti-HELFab antibody fragment was incorporated into E. coli in the same manner as in Example I, and subjected to SDS-PAGE. As a result of CBB staining and Western blotting, it was confirmed that the Fab gene was not expressed alone in the soluble fraction, but was all expressed in the precipitated fraction (FIG. 5).
- Mouse-derived anti-HEL scFv fragment gene was amplified by PCR. This gene was ligated to a T7 punoray vector by TA cloning, treated with NdeI / NotI, and then treated with the same restriction enzyme. By re-ligating to usion 2, a fusion protein expression system of TcFKBP18 and scFv was constructed.
- the obtained plasmid was incorporated into Escherichia coli in the same manner as in Example 7, and the transformant was obtained.
- This bacterium was cultured and recovered in the same manner as in Example 7, and stored frozen at 120 ° C.
- the obtained bacterial solution was subjected to SDS-PAGE in the same manner as in Example 7, and stained with CBB.
- Example 9 Including the mouse-derived anti-HEL scFv fragment prepared in Example 9.
- the DNA fragment treated with NdeI / NotI of the pT7 blue vector was ligated to TFf2 of Example 2 which had been treated with the same restriction enzymes in advance, so that the A fusion protein expression system was constructed.
- the obtained plasmid was incorporated into Escherichia coli in the same manner as in Example 7, and the transformant was obtained.
- This bacterium was cultured and collected in the same manner as in Example 7, and stored frozen at 120 ° C.
- the obtained bacterial solution was subjected to SD S_PAGE in the same manner as in Example 7, and stained with CBB.
- the fusion protein expression vector of TC FKB P18 and scFv prepared in Example 9 was treated with SpeI / NotI to prepare a DNA fragment containing the scFv fragment.
- the DNA fragment was ligated to the FK52 f2 of Example 3 previously treated with the same restriction enzymes to construct a fusion protein expression system of hFKBP52 and scFv.
- the obtained plasmid was integrated into Escherichia coli in the same manner as in Example 7, and the transformant was obtained.
- This bacterium was cultured and collected in the same manner as in Example 7, and stored frozen at 120 ° C.
- the obtained bacterial solution was subjected to SDS-PAGE in the same manner as in Example 7, and stained with CBB.
- the band density of the fusion protein expressed in the soluble fraction was measured with a densitometer, and was found to be about 9 ° / 0 of the total soluble protein derived from Escherichia coli.
- Example 11 CP 40 52 prepared in Example 4 was used instead of FK5 2 f 2 in Example 1. Re
- a fusion protein of hCyP40 and scFv was expressed in the same manner as in Example 11.
- the band density of the fusion protein expressed in the soluble fraction was measured with a densitometer, and as a result, was about 11% of the total soluble protein derived from Escherichia coli.
- HT la receptor human cello 1 and nin receptor
- a primer for PCR was designed based on the base sequence information registered as the NCB I code: HS S ERR 51, and the HTla receptor gene was obtained by PCR using a human cDNA library as type III. Was amplified.
- the amino acid sequence of HT1a is shown in SEQ ID NO: 13, and the nucleotide sequence is shown in SEQ ID NO: 14.
- the primer was provided with an Nde I restriction enzyme site on the 5 'side and a Not I restriction enzyme site on the 3' side. After introducing the PCR product into the pT7 blue T vector, it was confirmed that the sequence was not different from the registered information.
- the DNA fragment containing the HT1a gene was cleaved by NdeI / NotI treatment, purified, and ligated to FkpAf2 of Example 5 which had been previously treated with NdeIZNotI. The vector containing the HT1a gene was recovered.
- the obtained fusion protein expression vector of FkpA and HT1a was incorporated into Escherichia coli in the same manner as in Example 7, and the transformant was obtained.
- This bacterium was cultured and recovered in the same manner as in Example 7, and stored frozen at 120 ° C.
- the obtained bacterial solution was sonicated, centrifuged at 3000 rpm, and fractionated into a supernatant (soluble fraction) and a precipitate (precipitate fraction).
- the sample was subjected to SDS-PAGE in the same manner as in Example 7, and the expressed HT1a was specifically analyzed by staining with Coomassie brilliant blue (CBB) and Western blotting using an anti-serotoun receptor antibody. was detected.
- CBB Coomassie brilliant blue
- Example 13 Fusion of SurA and HT1a in the same manner as in Example 13 except that SurAf2 prepared in Example 6 was used instead of FkpAi2 in Example 13 The protein was expressed.
- CBB staining When subjected to SDS_PAGE in the same manner as in Example 7, staining with Coomassie Prienant Blue (CBB) and specifically detecting HT1a expressed by Western plotting using an anti-serotonin receptor antibody were performed.
- CB1 staining showed that HT1a was expressed in the soluble fraction in a form fused with SurA, and it was confirmed by Western blotting that it was indeed expressed.
- the band density of the fusion protein expressed in the soluble fraction was measured with a densitometer, and was found to be about 2% of the total soluble protein derived from E. coli.
- Example 9 The soluble fraction obtained in Example 9 was subjected to column purification in the following order of anion exchange chromatography and gel filtration in the following (a) and (b) to obtain a mouse-derived anti-HEL scFv. And the fusion protein of TcFKBP18 was purified to almost unity. The amount of fusion protein obtained as a result of the purification was about 50 mg / L of the medium.
- Solution B 25 mM HEPES-KOH buffer (pH 6.8) containing 0.5 M NaCl (0-300 min: solution B 0-100% linear gradient, 300-420 min: solution B 100%)
- the thrombin site of the fusion protein was cleaved by adding 10 U of thrombin to 1 mg of the fusion protein purified in Example 15 and treating at 22 ° C. for 16 hours. As a result of SDS-PAGE, it was confirmed that the fusion protein was indeed cleaved into mouse-derived anti-HELScFv and TcFKBP18 (FIG. 7).
- mice-derived anti-HEL scFv by ELISA Function of mouse-derived anti-HEL scFv by ELISA
- the function of mouse-derived anti-HELscFv obtained by expression was determined by the ELISA method using- ⁇ trilysozyme as an antigen. And whether it functions as a primary antibody. That is, 100 L of a 50 gZmL chicken egg white lysozyme (HEL) solution was added to a 96-well plate, and the plate was incubated at 30 ⁇ for 3 hours to immobilize HEL on the plate. After washing the plate with TBS buffer (pH 7.0), the plate was blocked with TBS buffer containing Block Ace (Dainippon Pharmaceutical) (4 ° C, overnight).
- TBS buffer pH 7.0
- TBS containing 10% Block Ace
- TBS containing 24 ⁇ g of mouse-derived anti-HEL scFv obtained in Example 16 was incubated at room temperature for 3 hours. After washing with TBS, the plate was incubated (2 hours, 30 ° C.) with a TBS buffer containing Anti_mouse IgG-HRP conjugate (Funakoshi) as a secondary antibody.
- TBS buffer containing Anti_mouse IgG-HRP conjugate (Funakoshi) as a secondary antibody.
- 100 zL of ABTS solution was added as a substrate for HRP, incubated for 30 minutes, and OD405 was measured.
- Fig. 8 shows the obtained results.
- the present invention Since the present invention has the above-mentioned constitution, it prevents the formation of inclusion bodies, which has been a problem in protein expression systems using bacteria, yeast, insect cells, and the like, and expresses a large amount of normal protein in a soluble fraction. It is possible to make. This eliminates the need to refold an inclusion body into a normal protein in vitro as in the related art.
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AT03733573T ATE458059T1 (de) | 2002-06-25 | 2003-06-25 | Expressionsvektor, wirt, fusionsprotein, verfahren zur herstellung von fusionsprotein und verfahren zur herstellung von protein |
DE60331311T DE60331311D1 (de) | 2002-06-25 | 2003-06-25 | Expressionsvektor, wirt, fusionsprotein, verfahren zur herstellung von fusionsprotein und verfahren zur herstellung von protein |
CA002490384A CA2490384A1 (en) | 2002-06-25 | 2003-06-25 | Expression vector, host, fused protein, process for producing fused protein and process for producing protein |
EP03733573A EP1516928B1 (en) | 2002-06-25 | 2003-06-25 | Expression vector, host, fused protein, process for producing fused protein and process for producing protein |
AU2003243969A AU2003243969B2 (en) | 2002-06-25 | 2003-06-25 | Expression vector, host, fused protein, process for producing fused protein and process for producing protein |
JP2004515182A JP4168028B2 (ja) | 2002-06-25 | 2003-06-25 | 発現ベクター、宿主、融合タンパク質、融合タンパク質の製造方法及びタンパク質の製造方法 |
US10/511,098 US20050130259A1 (en) | 2002-06-25 | 2003-06-25 | Expression vector, host, fused protein, process for producing fused protein and process for producing protein |
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Cited By (3)
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WO2005113768A1 (ja) * | 2004-05-21 | 2005-12-01 | Takara Bio Inc. | ポリペプチドの製造方法 |
JP2006230251A (ja) * | 2005-02-23 | 2006-09-07 | Kagoshima Univ | 融合タンパク質発現ベクター |
JP2010235492A (ja) * | 2009-03-31 | 2010-10-21 | Sekisui Chem Co Ltd | シャペロニン−目的タンパク質複合体及びその製造方法 |
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EP1971684B1 (en) | 2006-01-03 | 2011-10-05 | Roche Diagnostics GmbH | Chimaeric fusion protein with superior chaperone and folding activities |
AR086250A1 (es) | 2011-05-05 | 2013-11-27 | Hoffmann La Roche | Polipeptido de fusion presentador de una secuencia de aminoacidos y utilizacion del mismo |
US20190292535A1 (en) | 2012-11-08 | 2019-09-26 | Roche Diagnostics Operations, Inc. | Nucleic acids encoding chimeric polypeptides for library screening |
US10414818B2 (en) | 2013-09-27 | 2019-09-17 | Roche Diagnostics Operations, Inc. | Thermus thermophilus SlyD FKBP domain specific antibodies |
AU2016232771B2 (en) | 2015-03-18 | 2021-08-19 | Omnicyte | Fusion proteins comprising modified alpha virus surface glycoproteins and tumor associated antigen and methods thereof |
Citations (3)
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WO2000075346A1 (en) * | 1999-06-09 | 2000-12-14 | Medical Research Council | Fusion proteins comprising a fragment of a chaperon polypeptide |
JP2002262883A (ja) * | 2001-03-13 | 2002-09-17 | Sekisui Chem Co Ltd | モノクローナル抗体の製造方法 |
JP2002306182A (ja) * | 2000-05-26 | 2002-10-22 | Toyota Central Res & Dev Lab Inc | 抗菌蛋白質の製造方法、蛋白質融合体 |
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EP0871648A1 (en) * | 1995-09-15 | 1998-10-21 | Merck & Co., Inc. | A high throughput assay using fusion proteins |
US5989868A (en) * | 1997-09-12 | 1999-11-23 | The Board Of Regents Of The University Of Oklahoma | Fusion protein systems designed to increase soluble cytoplasmic expression of heterologous proteins in esherichia coli |
CA2344993C (en) * | 1998-10-02 | 2012-11-13 | Hansjorg Reimann | Method for the production of (poly)peptides by using truncated variants of the sv40 large t antigen with an intact n terminus |
JP4262594B2 (ja) * | 2001-06-22 | 2009-05-13 | エフ.ホフマン−ラ ロシュ アーゲー | レトロウイルス表面糖タンパク質を含む可溶性複合体 |
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- 2003-06-25 EP EP03733573A patent/EP1516928B1/en not_active Expired - Lifetime
- 2003-06-25 WO PCT/JP2003/008020 patent/WO2004001041A1/ja active Application Filing
- 2003-06-25 DE DE60331311T patent/DE60331311D1/de not_active Expired - Fee Related
- 2003-06-25 AT AT03733573T patent/ATE458059T1/de not_active IP Right Cessation
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WO2000075346A1 (en) * | 1999-06-09 | 2000-12-14 | Medical Research Council | Fusion proteins comprising a fragment of a chaperon polypeptide |
JP2002306182A (ja) * | 2000-05-26 | 2002-10-22 | Toyota Central Res & Dev Lab Inc | 抗菌蛋白質の製造方法、蛋白質融合体 |
JP2002262883A (ja) * | 2001-03-13 | 2002-09-17 | Sekisui Chem Co Ltd | モノクローナル抗体の製造方法 |
Non-Patent Citations (10)
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005113768A1 (ja) * | 2004-05-21 | 2005-12-01 | Takara Bio Inc. | ポリペプチドの製造方法 |
JPWO2005113768A1 (ja) * | 2004-05-21 | 2008-03-27 | タカラバイオ株式会社 | ポリペプチドの製造方法 |
JP4988337B2 (ja) * | 2004-05-21 | 2012-08-01 | タカラバイオ株式会社 | ポリペプチドの製造方法 |
JP2006230251A (ja) * | 2005-02-23 | 2006-09-07 | Kagoshima Univ | 融合タンパク質発現ベクター |
JP2010235492A (ja) * | 2009-03-31 | 2010-10-21 | Sekisui Chem Co Ltd | シャペロニン−目的タンパク質複合体及びその製造方法 |
Also Published As
Publication number | Publication date |
---|---|
CA2490384A1 (en) | 2003-12-31 |
US20050130259A1 (en) | 2005-06-16 |
EP1516928A1 (en) | 2005-03-23 |
ATE458059T1 (de) | 2010-03-15 |
DE60331311D1 (de) | 2010-04-01 |
JP4168028B2 (ja) | 2008-10-22 |
AU2003243969B2 (en) | 2008-03-13 |
EP1516928A4 (en) | 2006-11-08 |
JPWO2004001041A1 (ja) | 2005-10-20 |
EP1516928B1 (en) | 2010-02-17 |
AU2003243969A1 (en) | 2004-01-06 |
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