EP1141263A4 - Herstellung von löslichen rekombinanten trypsinanalogen - Google Patents
Herstellung von löslichen rekombinanten trypsinanalogenInfo
- Publication number
- EP1141263A4 EP1141263A4 EP99951445A EP99951445A EP1141263A4 EP 1141263 A4 EP1141263 A4 EP 1141263A4 EP 99951445 A EP99951445 A EP 99951445A EP 99951445 A EP99951445 A EP 99951445A EP 1141263 A4 EP1141263 A4 EP 1141263A4
- Authority
- EP
- European Patent Office
- Prior art keywords
- trypsinogen
- trypsin
- amino acid
- analog
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
Definitions
- the invention relates generally to recombinant DNA technology. More specifically, the present invention relates to recombinant trypsinogen analogs as well as methods for producing recombinant trypsin.
- Trypsin is a widely used serine protease which cleaves the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine.
- trypsin plays a pivotal role among pancreatic enzymes in the activation of endopeptidases.
- pancreatic enzymes are secreted through the pancreatic duct into the duodenum of the small intestine in response to a hormone signal generated when food passes from the stomach. They are not, however, synthesized in their final active form. Rather, they are made as slightly longer catalytically inactive molecules called zymogens.
- zymogens include trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidase. These zymogens must themselves be cleaved proteolytically to yield active enzymes.
- the first step of the activation cascade is the activation of trypsin from trypsinogen in the duodenum.
- Enteropeptidase also known as enterokinase
- enterokinase is a protease produced by duodenal epithelial cells which activates pancreatic trypsinogen to trypsin by excising a hexapeptide leader sequence from the amino-terminus of trypsinogen. Trypsin in turn autocatalytically activates more trypsinogen to trypsin and also acts on other proenzymes, thus, for example, liberating the endopeptidases chymotrypsin and elastase as well as carboxypeptidases A and B.
- This battery of enzymes work together with pepsin produced in the stomach and other proteases secreted by the intestinal wall cells to digest most ingested proteins into free amino acids, which can be absorbed by the intestinal epithelium.
- the enzymes themselves are continually subjected to autodigestion and other degradative processes so that high levels of these enzymes never accumulate in the intestine. In the pancreas, several factors oppose trypsinogen autoactivation, whereas in the duodenum, all the conditions favorable for typsinogen activation by enteropeptidase are present.
- a hexapeptide leader sequence which consists of Valine-Aspartate-Aspartate-Aspartate-Aspartate-Lysine ((Asp) 4 -Lys), on the amino-terminus of trypsinogen is enzymatically removed. Trypsinogens of many different species have been cloned and characterized. The pattern of (Asp) 4 -Lys at the amino-terminus, however, is well-conserved in all of these precursors. Mireaux Rovery, Limited Proteolyses in Pancreatic Chymotrypsinogens and Trypsinogens, 70 Biochimie 1131 (1988).
- Serine proteases such as trypsin have a variety of uses. They are useful for the characterization of other proteins as well as in the manufacturing process of other recombinant bioproducts. For example, small recombinant proteins are often expressed first as fusion proteins to facilitate their purification and enhance their stability.
- the fusion proteins can be engineered such that a leader sequence can be cleaved from the native protein sequence by trypsin. Any internal lysines or arginines that are not part of the leader sequence can be chemically protected from cleavage by trypsin.
- BSE bovine spongiform encephalopathy
- the present invention also provides an efficient and relatively inexpensive process to manufacture recombinant trypsin.
- the process of the present invention is significantly different from past approaches by providing for the expression of an inactive zymogen form that is soluble and properly folded yet is not activated until after purification from fermentation broth or cell extracts.
- This is accomplished through the expression of a single chain trypsinogen analog wherein the leader sequence is modified such that it lacks a trypsin-like enzyme cleavage site.
- the trypsinogen analogs of the present invention lack a lysine or arginine in the N-terminal leader sequence of the protein to prevent auto-activation or activation by endogenous host cell enzymes. Once expressed in a particular cell type, the trypsinogen analog can then be secreted outside of the cell and isolated from the culture medium or alternatively expressed in the cytoplasm.
- the cells can be collected by centrifugation, lysed, and the trypsinogen isolated. Once the trypsinogen is isolated, stability can be enhanced by lowering the pH to about 3.0 and then subsequently activating the trypsinogen with an aminopeptidase such as dipetidylaminopeptidase (DAP).
- DAP dipetidylaminopeptidase
- the present invention provides trypsinogen analogs which have a modified amino- terminal leader sequence which results in recombinant trypsinogen that is stable and easy to activate.
- This invention provides a means to move away from animal-sourced trypsin and avoid the problems of degradation, instability, and damage to cell membranes which occurs during expression and/or secretion of recombinantly produced trypsinogen.
- the invention makes possible the secretion of a folded trypsinogen, therefore eliminating costly and time consuming refolding steps.
- the present invention provides a trypsinogen analog comprising a protein having trypsin activity fused to a leader sequence having at least 2 amino acids wherein the amino acids are any amino acid except Lys or Arg.
- the invention provides a trypsinogen analog represented by X-AA-Y, wherein Y is a protein having trypsin activity, AA is an amino acid other than Lys or Arg and X-AA is a leader sequence having at least 2 amino acids.
- the invention further provides analogs that can be activated by cleavage with an aminopeptidase.
- the mature trypsin protein, which is the active enzyme is derived from a single polypeptide which comprises a leader sequence fused to a polypeptide representing the active enzyme.
- the leader sequence must be cleaved to generate the active trypsin molecule. Still further, the invention provides an isolated trypsinogen analog represented by X-AA-Y, wherein: Y is a protein having trypsin activity; AA is any amino acid except Lys or Arg; and X-AA is a leader sequence having at least 2 amino acids.
- the invention encompasses leader sequences fused to trypsin which cannot be cleaved by trypsin, enteropeptidases, or any other endogenous trypsin-like enzymes that may be present during expression, secretion, or isolation of the protein.
- the leader sequence of the trypsinogen analog is selected from the group consisting of: ) from 2 to 20 non basic amino acid residues; b) from 2 to 20 amino acid residues wherein the amino acid residues can be any residue except except Lys or Arg; c ) Val-Asp-Asp-Asp-Asp-N, wherein N is any amino acid residue except Lys, Arg, or Pro; d) amino acid residues 5 through 10 of SEQ ID NO:2; e ) from 2 to 20 amino acid residues wherein the residues can be any hydrophilic amino acid residue that is not Lys or Arg; f ) amino acid residues 5 through 8 of SEQ ID NO:2; and g) from 2 to 20 amino acid residues, wherein the number of residues is an even number of residues and the residues are any non-basic amino acid except proline.
- this invention provides an inactive trypsin precursor comprising the sequence which is SEQ ID NO:2.
- the invention also provides proteins having the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO 4, including derivatives without the first three amino acids of SEQ ID NO: 2 and the first two amino acids of SEQ ID NO 4.
- the invention further provides a trypsinogen analog having a secretion signal sequence of amino acids operably linked to the amino-terminal end of the leader sequence wherein said signal sequence allows the trypsinogen to be secreted into the host cell growth medium.
- the invention encompasses both RNA and DNA which encode the trypsinogen analog proteins of the present invention.
- Preferred polynucleotides of the current invention include the nucleic acids of SEQ ID NO:l and SEQ ID NO:3 and variants thereof.
- This invention also provides recombinant vectors comprising the above-described nucleic acids as well as host cells harboring said recombinant vectors.
- the invention further provides essentially chymotrypsin-free trypsin preparations.
- the invention also provides nucleic acids sharing at least about 65 % identity with the nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 3.
- the invention includes methods for making recombinant trypsinogen analogs comprising the steps of: expressing the trypsinogen analogs described above in a host cell and isolating the expressed trypsinogen. Methods are also provided for making recombinant trypsin further comprising the step of activating the isolated trypsinogen with an aminopeptidase.
- methods comprising the steps of: expressing trypsinogen analogs in Pichia pastoris; isolating the trypsinogen analogs in a buffer; and incubating the isolated trypsinogen with a dipeptidylaminopeptidase in a buffer with a pH from about 2.0 to about 6.5 such that the trypsinogen is converted to active trypsin.
- Figure 1 is a restriction map of the plasmid vector pRMG5 containing the bovine trypsin gene.
- Figure 2 is a restriction map of the plasmid vector pLGD43 containing the bovine trypsin gene.
- FIG. 3 summarizes the production of ValAsp5 trypsinogen in the two expression systems described herein.
- Base pair refers to DNA or RNA.
- the abbreviations A,C,G, and T correspond to the 5'-monophosphate forms of the deoxyribonucleosides (deoxy)adenosine, (deoxy)cytidine, (deoxy)guanosine, and thymidine, respectively, when they occur in DNA molecules.
- the abbreviations U,C,G, and A correspond to the 5'- monophosphate forms of the ribonucleosides uridine, cytidine, guanosine, and adenosine, respectively when they occur in RNA molecules.
- base pair may refer to a partnership of A with T or C with G.
- heteroduplex base pair may refer to a partnership of A with U or C with G. (See the definition of "complementary”, infra.)
- the terms "digestion” or “restriction” of DNA refers to the catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA (“sequence-specific endonucleases”).
- restriction enzymes used herein are commercially available and their reaction conditions, cofactors, and other requirements were used as would be known to one of ordinary skill in the art. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer or can be readily found in the literature.
- Ligation refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments. Unless otherwise provided, ligation may be accomplished using known buffers and conditions with a DNA ligase, such as T4 DNA ligase.
- Plasmid refers to an extrachromosomal (usually) self-replicating genetic element. Plasmids are generally designated by a lower case “p” followed by letters and/or numbers.
- the starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accordance with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
- reading frame means the nucleotide sequence from which translation occurs “read” in triplets by the translational apparatus of transfer RNA (tRNA) and ribosomes and associated factors, each triplet corresponding to a particular amino acid.
- tRNA transfer RNA
- ribosomes and associated factors each triplet corresponding to a particular amino acid.
- the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame” being maintained.
- Recombinant DNA cloning vector refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
- recombinant DNA expression vector refers to any recombinant DNA cloning vector in which a promoter to control transcription of the inserted DNA has been incorporated.
- expression vector system refers to a recombinant DNA expression vector in combination with one or more trans-acting factors that specifically influence transcription, stability, or replication of the recombinant DNA expression vector.
- the trans-acting factor may be expressed from a co-transfected plasmid, virus, or other extrachromosomal element, or may be expressed from a gene integrated within the chromosome.
- Transcription refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
- transfection refers to the taking up of an expression vector by a host cell whether or not any coding sequences are in fact expressed. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, calcium phosphate co-precipitation, and electroporation. Successful transfection is generally recognized when any indication of the operation of this vector occurs within the host cell.
- transformation means the introduction of DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.
- Methods of transforming bacterial and eukaryotic hosts are well known in the art, many of which methods, such as nuclear injection, protoplast fusion or by calcium treatment using calcium chloride are summarized in J. Sambrook, et al.. Molecular Cloning: A Laboratory Manual, (1989). Generally, when introducing DNA into Yeast the term transformation is used as opposed to the term transfection.
- translation refers to the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
- vector refers to a nucleic acid compound used for the transfection and/or transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transfected and/or transformed. Plasmids, viruses, and bacteriophage are suitable vectors. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases.
- vector as used herein includes Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
- complementarity refers to pairs of bases (purines and pyrimidines) that associate through hydrogen bonding in a double stranded nucleic acid.
- bases purines and pyrimidines
- the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil.
- hybridization refers to a process in which a strand of nucleic acid joins with a complementary strand through base pairing.
- the conditions employed in the hybridization of two non-identical, but very similar, complementary nucleic acids varies with the degree of complementarity of the two strands and the length of the strands. Such techniques and conditions are well known to practitioners in this field.
- isolated amino acid sequence refers to any amino acid sequence, however constructed or synthesized, which is locationally distinct from the naturally occurring sequence.
- isolated DNA compound refers to any DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location in genomic DNA.
- isolated nucleic acid compound refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.
- a “primer” is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation.
- promoter refers to a DNA sequence which directs transcription of DNA to RNA.
- a “probe” as used herein is a nucleic acid compound or a fragment thereof which hybridizes with another nucleic acid compound.
- stringency refers to a set of hybridization conditions which may be varied in order to vary the degree of nucleic acid affinity for other nucleic acid. (See the definition of "hybridization”, supra.)
- PCR refers to the widely-known polymerase chain reaction employing a thermally-stable DNA polymerase.
- leader sequence refers to a sequence of amino acids which can be enzymatically or chemically removed to produce the desired polypeptide of interest.
- processed polypeptide refers to a polypeptide or protein wherein the N- terminal leader sequence has been removed to yield the desired polypeptide of interest.
- secretion signal sequence refers to a sequence of amino acids generally present at the N-terminal region of a larger polypeptide functioning to initiate association of that polypeptide with the cell membrane and secretion of that polypeptide through the cell membrane.
- trypsinogen refers to an inactive serine protease zymogen which can be converted to trypsin by removal of a hexapeptide leader sequence which generally comprises the sequence Val(Asp) 4 Lys (SEQ ID NO:?).
- trypsin or "trypsin-like enzymes” refer to proteases which have the ability to cleave the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine.
- enterokinase or "enteropeptidase” refer to proteases generally produced in epithelial cells that activate trypsinogn to trypsin by cleaving off the trypsinogen leader sequence from the amino terminus of the protein.
- trypsinogen analog refers to trypsinogen which has been mutated such that it cannot be converted to active trypsin by the action of trypsin or trypsin-like enzymes.
- autoactivation or “autocatalytic” refers to the ability of trypsin to activate trypsinogen by cleaving the leader sequence to produce more active trypsin.
- endogenous trypsin-like enzyme refers to proteases which have the ability to cleave the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine and which are normally expressed in the particular cell type of interest.
- percent identity is used with reference to the Blast 2 algorithm, which is available at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST), using default parameters. References pertaining to this algorithm include: those found at http://www.ncbi.nlm.nih.gov/BLAST/T3last_references.html; Altschul, S.F., Gish, W., Miller, W., Myers, E. W. & Lipman, D.J. (1990) "Basic local alignment search tool.” J. Mol. Biol. 215:403-410; Gish, W. & States, D.J.
- the present invention avoids the premature activation of recombinant trypsinogen to trypsin that can occur during expression, secretion, and isolation.
- Prior experimentation involving recombinant trypsinogen indicates that it is generally quite difficult to provide a process for producing stable recombinant trypsinogen.
- Attempts by researchers experimenting in the field of recombinant protein expression to make recombinant trypsin were not successful, in part, because a mixture of different species of active and inactive trypsin was produced.
- the final product was not fully active mature recombinant trypsin. Trypsin contains three internal trypsin cleavage sites in addition to the cleavage site in the leader sequence.
- Trypsin has a strong affinity for itself. Prior processes were problematic because, during expression and/or secretion, trypsinogen was activated to trypsin by endogenous enzymes. These activated trypsin molecules then could cleave other recombinantly produced trypsin enzymes at internal cleavage sites and render these enzymes inactive. The resulting mixture of recombinant trypsin peptides contained only a small percentage of intact active trypsin. In addition, activation of trypsin during expression or secretion can damage the cell membrane of the host. This can also contribute to low yields of recombinant trypsinogen or trypsin.
- trypsinogen analogs described herein circumvent the premature activation problem because they are mutated at the activation site to prevent autoactivation or activation by endogenous trypsin-like enzymes.
- classical activation of trypsinogen into trypsin by enterokinase or trypsin would not be possible.
- Trypsin proteins having trypsin activity have been exceptionally good subjects for a molecular analysis of protein structure and function. Trypsin proteins have been isolated and characterized from numerous species including bovine, rat, and humans. Le Huerou et al. (1990) Isolation and nucleotide sequence ofcDNA clone for bovine pancreatic anionic trypsinogen. Structural identity within the trypsin family, Eur. J. Biochem. 193, 767-773; Craik, C.S. et al. (1984) J.Biol. Chem. 259:14255-14264; Fletcher, T.S. et al. (1987) Biochemistry 26:3081-3086.
- a protein having trypsin activity includes a large group of enzymes which are well-conserved between species and which function by cleaving the peptide bond on the carboxy-terminus of basic amino acid residues such as lysine and arginine.
- Wild-type trypsinogens generally contain a hexapeptide leader sequence consisting of Val-Asp-Asp-Asp-Asp-Lys. Trypsin and endogenous trypsin-like enzymes generally cleave the C-terminal peptide bond of Arg and Lys.
- the present invention provides trypsinogens that have modified leader sequences which maintain the molecule as an inactive zymogen that cannot be activated by trypsin-like enzymes.
- leader sequence may be used in place of the native leader peptide to prevent activation by tryspin-like enzymes.
- the number of amino acids in the N-terminal leader sequence can be as small as two amino acids which will maintain the enzyme in an inactive state. Amino acid chains longer than six are also possible. N-terminal leader sequences, however, longer than about 20 amino acids slow down the subsequent aminopeptidase activation process.
- the leader sequence be between four and six amino acids in length to maintain inactivity and provide stability while at the same time allowing efficient subsequent activation by aminopeptidases.
- an odd number of amino acid residues making up the leader sequence will not generally be processed cleanly by DAP.
- the leader sequence constitute four or six amino acids if DAP is to be used as the activating aminopeptidase.
- the present invention also contemplates leader sequences having a variable sequence of amino acids.
- the leader sequence is preferably exposed to the solvent which enables it to be cleaved by DAP or other aminopeptidases.
- a leader sequence consisting of amino acids which will facilitate exposure of the leader to the solvent and allow for subsequent removal by DAP is preferred.
- a preferred leader sequence will contain an even number from two to twenty hydrophilic amino acids which cannot be cleaved by trypsin-like enzymes, but which can be removed by DAP or other aminopeptidases.
- a more preferred leader sequence provides a single amino acid substitution in the native trypsinogen leader sequence wherein the first lysine encountered, for example, position 6 of the bovine typrsinogen zymogen, is substituted with any amino acid except Arg or Pro.
- An even more preferred leader sequence comprises the sequence Val-Asp-Asp-Asp-Asp- Asp (amino acids 5 through 10 of SEQ ID NO:2) immediately N-terminal to the first amino acid of the mature active trypsin enzyme.
- Wild-type trypsinogen genes can be obtained by a plurality of recombinant DNA techniques including, for example, hybridization, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis.(5ee e.g., T. MANIATIS ET AL., MOLECULAR CLONING: A LABORATORY MANUAL, (2d ed. 1989). The isolated gene can then be modified or mutated to encode the trypsinogen analogs described above.
- PCR polymerase chain reaction
- the isolated nucleic acids of the present invention can be prepared by direct chemical synthesis by methods such as the phosphotriester method of Narang, et al., Meth. Enzymol. 68:90-99 (1979); the phosphodiester method of Brown, et al., Meth. Enzymol.68: 109-151 (1979); the diethylphosphoramiditemethod of Beaucage, et al., Tetra. Letts.22:1859-1862 ( 1981 ); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra.
- the trypsinogen cDNA can be isolated from a library constructed from any tissue in which said gene is expressed.
- Methods for constructing cDNA libraries in a suitable vector such as a plasmid or phage for propagation in prokaryotic or eukaryotic cells are well known to those skilled in the art. (See e.g., MANIATIS ET AL., supra). Suitable cloning vectors are well known and are widely available.
- mRNA is isolated from a suitable tissue, and first strand cDNA synthesis is carried out.
- a second round of DNA synthesis can be carried out for the production of the second strand.
- the double-stranded cDNA can be cloned into any suitable vector, for example, a plasmid, thereby forming a cDNA library.
- a variety of different cDNA libraries can be purchased commercially (Clontech Laboratories Inc., Palo Alto, California).
- Oligonucleotide primers targeted to any suitable region of the trypsinogen gene can be used for PCR amplification. See e.g. PCR PROTOCOLS: A GUIDE TO METHOD AND APPLICATION (M. Innis et al. eds., 1990).
- the PCR amplification comprises template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT). A positive result is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis.
- An object of the present invention is to provide a process whereby the trypsinogen analogs described herein can be expressed by recombinant methods. Recombinant protein expression is preferred to obtain a high yield of highly pure protein, especially when the goal is to use these proteins in the manufacturing process for other therapeutic biomolecules.
- the basic steps in the recombinant production of desired proteins are: a ) construction of a synthetic or semi-synthetic DNA encoding the protein of interest; b ) integrating said DNA into an expression vector in a manner suitable for the expression of the protein of interest, either alone or as a fusion protein; c ) transforming an appropriate eukaryotic or prokaryotic host cell with said expression vector, d) culturing said transformed or transfected host cell in a manner to express the protein of interest; and e ) recovering and purifying the recombinantly produced protein of interest.
- the invention further provides trypsinogen-encoding nucleic acids. These are exemplified by the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3, and their complements. Also included in the inventive nucleic acids are those closely related to the sequences of SED ID NO: 1 and SED ID NO: 3, yet retain the ability to be activated to form mature trypsin. Generally, these sequences share at least about 65 percent identity with SEQ ID NOS. 1 .and 2, but more typically share at least about 70 percent identity. More preferred embodiments share at least about 75% identity, and some share at least about 80% identity. Even more preferred nucleic acids share at least about 85% identity or at least about 90% identity. Most preferred nucleic acids share at least about 95% identity, with some sharing at least about 98 percent or 99 percent identity.
- the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of trypsinogen analog polypeptides or fragments thereof by recombinant techniques.
- the nucleotides encoding trypsinogen analogs can optionally be joined to a vector containing a selectable marker for propagation in a host.
- a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid, or by other methods that are well known to those with ordinary skill in the art.
- the vector is a viral vector, it can be introduced directly into mammalian host cells or introduced using viral supernatant produced by packaging in vitro using an appropriate packaging cell line.
- Bacterial viral vectors can also be packaged in vitro using packaging cell extracts commercially available and then tranfected into host bacterial cells.
- the DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, as well as the glyceraldehyde phosphate dehydrogenase (GAPDH) and alcohol oxidase (AOX) promoters to name a few. Other suitable promoters will be known to the skilled artisan.
- the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
- the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated.
- Expression vectors will preferably include at least one selectable marker.
- markers include, e.g., dihydrofolate reductase or neomycin resistance for mammalian cell culture, neomycin resistance or complementation of auxotrophic markers for Yeasts, and tetracycline, ampicillin, kanamycin, or chloramphenicol resistance genes for culturing in E. coli and other bacteria.
- Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
- coli Streptomyces and Salmonella typhimurium cells
- fungal cells such as Aspergillus niger
- yeast cells such as Pichia pastoris and Saccharomyces cerevisiae
- insect cells such as Drosophila S2 and Spodoptera Sf9 cells
- animal cells such as CHO, COS and Bowes melanoma cells
- plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
- Vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
- Preferred eucaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
- Preferred vectors for expression in Pichia pastoris include pLDG vectors (figure 1) and pPIC vectors commercially available from In vitrogen, Inc. Other suitable vectors will be readily apparent to the skilled artisan.
- Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, transformation or other methods. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
- Trypsinogen analogs of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids can be added to the N-terminus of an analog to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to facilitate purification. Such regions can be removed prior to final preparation of an active enzyme. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.
- nucleic acids of the present invention may express a protein of the present invention in a recombinantly engineered cell, such as bacteria, yeast, insect, or mammalian cells.
- a recombinantly engineered cell such as bacteria, yeast, insect, or mammalian cells.
- the cells produce the protein in a non-natural condition (e.g., in quantity, composition, location, and/or time), because they have been genetically altered through human intervention to do so.
- the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression vector.
- the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
- Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention.
- expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translationterminator.
- modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to facilitate purification of the protein or other cleavages to create conveniently located restriction sites or termination codons.
- additional amino acids e.g., poly His
- nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding a trypsinogen analog of the present invention.
- Such methods are well known in the art, e.g., as described in US patentNos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
- Prokaryotic cells may be used as hosts for expression. Prokaryotes most frequently are represented by various strains of E. coli; however, other microbial strains may also be used. Commonly used prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta lactamase (penicillinase)and lactose (lac) promoter systems (Chang, et al., Nature 198:1056 (1977)), the tryptophan (trp) promoter system (Goeddel, et al., Nucleic Acids Res.
- bacteriaphage T7 promoter and RNA polymerase the bacteriaphage T7 promoter and RNA polymerase, and the bacteriaphage lambda derived P L promoter and N-gene ribosome binding site (Shimatake, et al., Nature 292: 128 (1981)).
- selection markers include genes specifying resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol.
- Bacterial vectors are typically of plasmid or phage origin. Appropriate bacterial cells are infected with phage vector particles or transfected with naked phage vector DNA. If a plasmid vector is used, the bacterial cells are transformed with the plasmid vector DNA. Expression systems for expressing a protein of the present invention are available using Bacillus sp. and Salmonella (Palva, et al., Gene 22:229-235 (1983); Mosbach, et al., Nature 302:543-545 (1983)).
- eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art. As explained briefly below, a nucleic acid of the present invention can be expressed in these eukaryotic systems.
- yeast Synthesis of heterologous proteins in yeast is well known.
- F. Sherman, et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982) is a well-recognized work describing the various methods available to produce the protein in yeast.
- Two widely utilized yeast for production of eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.
- Vectors, strains, and protocols for expression in Saccharomyces and Pichia are known in the art and available from commercial suppliers (e.g., Invitrogen).
- Suitable vectors usually have expression control sequences, such as promoters, including 3-phosphoglyceratekinase or alcohol oxidase, and an origin of replication, termination sequences and the like as desired.
- sequences encoding proteins of the present invention can also be ligated to various expression vectors for use in transfecting cell cultures of, for instance, mammalian, insect, or plant origin.
- Illustrative of cell cultures useful for the production of the peptides are mammalian cells. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used.
- a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21 , and CHO cell lines.
- Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk (phosphoglyceratekinase) promoter), an enhancer (Queen, et al., Immunol. Rev. 89:49 ( 1986)), and processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
- Other animal cells useful for production of proteins of the present invention are available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (7th edition, 1992).
- Appropriate vectors for expressing proteins of the present invention in insect cells are usually derived from the SF9 baculovirus.
- suitable insect cell lines include mosquito larvae, silkworm, army worm, moth and Drosophila cell lines such as a Schneider cell line (See Schneider, J. Embryol. Exp. Morphol.27:353-365 (1987).
- polyadenylation or transcription terminator sequences are typically incorporated into the vector.
- An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
- An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol.45:773-781 ( 1983)).
- gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus type- vectors. M.
- Signal peptides may be used to facilitate the extracellular discharge of proteins in both prokaryotic and eukaryotic environments. It has been shown that the addition of a heterologous signal peptide to a normally cytosolic protein may result in the extracellular transport of the normally cytosolic protein. Alternate signal peptide sequences may function with heterologous coding sequences.
- Signal peptides are well known in the art and can be incorporated into the modified trypsinogen structure to facilitate extracellular translocation or intracellular destination.
- the signal peptide used is a signal peptide native to a secretory protein of the host cell line.
- the signal peptide is the alpha factor signal sequence. This signal sequence is fused to the N-terminal end of the trypsinogen analog leader sequence and will result in the extracellular transport of trypsinogen analogs expressed in Yeast.
- HSA human serum albumin
- an expression vector carrying the trypsinogen analog gene is transfected into a suitable host cell using standard methods, cells that contain the vector are propagated under conditions suitable for expression of the recombinant trypsinogen analog protein.
- suitable growth conditions would incorporate the appropriate inducer.
- the recombinantly-produced protein may be purified from cellular extracts of transformed cells by any suitable means.
- Trypsinogen analogs of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, reversed-phase chromatography, hydroxylapatite chromatography, and lectin chromatography. Most preferably, ultra-filtration is employed coupled with cation exchange chromatography.
- the trypsinogen analog may be fused at its N-terminal end to several histidine residues.
- This "histidine tag” enables a single-step protein purification method referred to as “immobilized metal ion affinity chromatography” (IMAC), essentially as described in U.S. Patent 4,569,794, which hereby is incorporated by reference.
- IMAC immobilized metal ion affinity chromatography
- trypsinogen analog which has been secreted into the culture medium is concentrated by ammonium sulfate precipitation (70%).
- the precipitate is resuspended in 20 mM sodium acetate, pH 5.0 and dialyzed against the same buffer.
- the trypsinogen analog can then be loaded on a CM-Sepharose CL6B Column (Pharmacia, Upsala, Sweden) and eluted with a gradient of NaCl.
- An alternative purification process from cell culture growth medium begins with centrifugation of the growth medium to pellet cells.
- the pH of the supernatant is adjusted to about 3.0 preferably with an acetate buffer.
- the adjusted supernatant is then subjected to ultra-filtration (tangential flow filtration with a 300 kDa/ 10 kDa molecular weight cutoffs).
- the filtrate can optionally be further purified using cation exchange chromatography.
- the purified isolate then is processed using an aminopeptidase such as DAP or MAP.
- the reaction is then concentrated again using ultrafiltration resulting in yields around 50%.
- the present invention provides a process for activating the trypsinogen analog following expression of the trypsinogen in bacteria, yeast, or higher eukaryotic cells. Following digestion with an aminopeptidase such as a mono- or diaminopeptidase, an active trypsin protein with an N-terminus identical to that of wild type trypsin can be obtained. The conformation of trypsinogen will prevent aminopeptidases from cleaving beyond the first amino acid of the mature trypsin molecule. Numerous aminopeptidases exist and can be used to activate the trypsinogen analogs of the present invention. Watson et al. (1976) Methods Microb. 9:1-14 describe different aminopeptidases present in different bacteria including E. coli and is entirely herein incorporated by reference.
- the trypsinogen Before processing the trypsinogen analogs using an aminopeptidase, however, the trypsinogen may be purified from a cell lysate or, in the case of a secreted protein, from the culture medium. A variety of purification steps may be employed including ammonium sulfate precipitation followed by dialysis and column chromatography.
- DAP dipeptidylaminopeptidase
- Dipeptidylaminopeptidases are enzymes which hydrolyze the penultimate amino terminal peptide bond releasing dipeptides from the unblocked amino-termini of peptides and proteins.
- There are currently four classes of dipeptidylaminopeptidases (designated DAP-I, DAP-II, DAP-III and DAP-IV) that differ based on their physical characteristics and the rates at which they react with their substrates.
- DAP I is a relatively non-specific DAP that catalyzes the release of many dipeptide combinations from the unblocked amino termini of peptides and proteins.
- DAP I shows little or no activity if the emergent dipeptide is Pro-X, or X-Pro (where X is any amino acid).
- DAP II shows a preference for amino terminal dipeptide sequences that begin with Arg-X or Lys-X, and to a lesser extent, X-Pro.
- DAP-II exhibits significantly lower reaction rates versus most other dipeptide combinations.
- DAP III appears to have a propensity toward amino terminal dipeptide sequences of the form Arg- Arg and Lys-Lys.
- DAP IV shows its highest rate of hydro lytic activity toward dipeptide sequences of the form X-Pro.
- the DAP enzymes, particularly DAP-I and DAP-IV have been shown to be useful in processing proteins.
- leader sequence present on any protein molecule could be removed by an aminopeptidase as long as the secondary structure of the protein was such that the peptidase could not cleave beyond the first critical residue of the desired end product protein.
- the activation reaction can be carefully controlled to ensure that cleavage does not occur beyond the first amino acid of the mature protein.
- the DAP reaction that converts inactive trypsinogen analogs into active trypsin is generally conducted in an aqueous medium suitably buffered to obtain and maintain a pH from about 2.0 to about 6.5.
- the pH of the medium ranges from about 3.0 to about 4.5, and most preferably, from about 3.0 to about 3.5.
- dDAP to remove dipeptides from trypsinogen is advantageous because dDAP's pH optimum of 3.5 allows the reaction to be run at acidic pH.
- a solubilizing agent such as urea, sodium dodecylsulfate, guanidine, and the like, may be employed.
- Another product of the invention is a highly purified preparation of trypsin.
- Trypsin is defined below.
- the present trypsin is produced in a recombinant system that results in secretion into the medium, it is anticipated that the resulting preparation should by highly purified and have negligible levels of contaminating chymotrypsin activity.
- This highly purified trypsin meets at least one of the following criteria of purity. Generally such preparations are greater than about 90 percent pure. More typically, however, these preparations are more than about 95 percent pure and preferably they are at least about 99 percent pure, and generally at least about 99.5, 99.9 or 99.99 percent pure. Some preparations have no detectable contaminants.
- Purity may also be evaluated by reverse phase high performance liquid chromatography method as presented generally below in the Examples. In such a case, percent purity is evaluated by comparing the integration of the trypsin peak to all peaks.
- the preparations of the invention generally show no chymotrypsin by this method.
- Purity may also be as measured by specific activity using the TAME assay, detailed below. Reference pure material should yield about 235 U/mg.
- preferred trypsin preparations are "essentially chymotrypsin-free.”
- "essentially chymotrypsin-free” denotes a preparation that has less than about 0.01% chymotrypsin (by weight, relative to trypsin). More preferred essentially chymotrypsin-free preparations generally have less than about 0.005%, and even more preferred preparations have less than about 0.001% chymotrypsin and most preferred preparations have less than about 0.0005% chymotrypsin.
- Some highly purified preparations are expected to meet the foregoing criteria of purity when measured using the ultra-sensitive glucagon-based high performance liquid chromatography (HPLC) method presented below in Example 6. In such an assay, preferred compositions should show no detectable chymotryptic glucagon peaks.
- the trypsin preparations of the invention also are free of any cellular components derive from Aspergillus, or related organisms, such as proteins and toxins.
- the trypsin preparations of the invention generally are essentially mammalian protein-free.
- essentially mammalian protein-free compositions refer to any of the inventive compositions that are essentially free of all mammalian proteins, except, of course, for the recombinantly-produced product. In general, this is achieved by avoiding the addition of mammalian proteins, like casein (added to bacterial cultures), and producing the proteins in a non-mammalian host. Excluding the recombinant protein, typical compositions have less than about 1% mammalian protein, but usually have less than about 0.5% mammalian protein or less than about 0.1% mammalian protein.
- compositions have less than about 0.01% mammalian protein and most preferred compositions have no detectable mammalian protein.
- ELISAs enzyme-linked immunosorbant assays
- the essentially chymotrypsin-free trypsin of the invention may be assembled into "commercial units" that are suitable for sale.
- Each commercial unit generally comprises a bulk quantity of essentially chymotrypsin-free trypsin.
- a bulk quantity usually comprises at least about 10 mg of essentially chymotrypsin-free trypsin unit. More typically, however, larger quantities will be present, such as at least about 50 mg per unit, at least about 100 mg per unit, at least about 500 mg per unit or at least about 1 gram per unit. Even larger quantities, e.g., a. unit of at least about a fifty grams, a unit of at least about a hundred grams unit, or a unit of at least about a kilogram(s), also are contemplated.
- commercial unit also contemplates assemblages of smaller commercial units to form larger ones.
- a one kilogram commercial unit of essentially chymotrypsin-free trypsin may be provided as a thousand one-gram commercial units.
- a commercial unit will contain essentially chymotrypsin-free trypsin as a liquid solution. It may, however, be present in a solid form, such as a freeze-dried (lyophylized) powder. Bulking agents and stabilizers (like calcium) are optionally included.
- a commercial unit also includes the packaging containing the essentially chymotrypsin-free trypsin, and optionally includes printed product specifications, an inventory control number and/or instructions for use.
- Wild type bovine trypsinogen was mutated to destroy the trypsin cleavage site.
- the Lys residue present in the leader sequence of the native bovine trypsinogen protein was mutated to an Asp residue (position 10 of SEQ ID NO:2).
- At the DNA level the mutation generates an EcoRV site which is useful for subsequent modifications of different constructs. All constructs were first assembled using the vector pRMG5 (Fig. 1) and then transferred to Pichia pastoris expression vectors.
- the expression vector pPIC9 supplied by In Vitrogen was used to create a trypsinogen analog fused to the ⁇ -factor signal sequence.
- the DNA encoding the fusion protein was cloned downstream of the methanol inducible AOX1 promoter.
- the pPIC9 vector contains the AOX 1 promoter cloned 5' to the ⁇ -factor signal sequence which in turn is cloned 5' to a multiple cloning sequence.
- the vector was constructed such that DNA encoding a Glu-Ala-Glu-Ala (amino acids 1 through 4 of S ⁇ Q ID NO:2) peptide was inserted between the C-terminus of the alpha factor signal sequence and the N-terminus of the trypsinogen analog leader sequence to improve the yield of secreted protein.
- oligonucleotide final concentrations were about 2.5 ⁇ g/ ⁇ l.
- each oligonucleotide was phosphorylated with 4 Units of T4 polynucleotide kinase for 1 hour at 37°C in a buffer containing 6 mM MgCl2, 6 mM DTT, 0.6 mM ATP and 120 mM Tris, pH 8.0.
- the phosphorylated oligonucleotides were then pooled and annealed.
- the resulting double stranded oligonucleotides were then cloned into pRMG5 to create pRMG5- ⁇ F( ⁇ A) 2 VD5.
- the pRMG5 vector was prepared by digesting the vector with Xh ⁇ l and N ⁇ rl. After digestion, the vector was dephosphorylated with 0.1 U of calf intestinal phosphatase (CIP) at 37°C for 30 minutes. The vector was then ligated to the double stranded phosphorylated oligonucleotides. The resulting clone was used for subcloning the trypsinogen analog in pPIC9.
- the pPIC9 expression vector was prepared in the dam minus E.coli GM82 strain and digested with vrll (Xbal cohesive) and Xhol and then dephosphorylated with 0.1 U of CIP.
- the trypsinogen analog was extracted from pRMG5- ⁇ F(EA) 2 VD5 as a XhollXbal fragment and subcloned into pPIC9.
- the resulting plasmid, pPIC9- ⁇ F(EA) 2 VD5 contained DNA encoding the alpha factor signal sequence ( ⁇ F) fused to the Glu-Ala-Glu-Ala ((EA) 2 ) insertion fused to the leader Val(Asp) 5 (VD5) fused to bovine trypsin.
- Trypsinogen was also fused directly to the C-terminus of the alpha factor without the (GluAla) 2 insertion.
- Two oligonucleotides, 5'-TC GAG AAA AGA GTC GAC GAT GAT GAC GAT ATC GTT GGA GTT TAT ACA TGT GG-3' and 5'-CGC CAC ATG TAT AAC CTC CAA CGA TAT CGT CAT CAT CGT CGA CTC TTT TC-3' encoding a C-terminal portion of the alpha factor signal sequence and the Val(Asp) leader sequence were synthesized, prepared as described above, and ligated into the Xhol and N ⁇ rl sites of pRMG5.
- a positive clone with the correct sequence was then used to subclone the trypsinogen gene into pPIC9 as described above.
- the resulting clone was named pPIC9- ⁇ FVD5 and contained D ⁇ A encoding the alpha factor signal sequence fused to the leader Val(Asp) 5 fused to bovine trypsin.
- the trypsinogen analog gene is under the control of the glyceraldehyde phosphate dehydrogenase (GAPDH) promoter and the HSA secretion signal sequence is used to direct the protein to the outside of the cell.
- GPDH glyceraldehyde phosphate dehydrogenase
- the cloning strategy involves first ligating a trypsinogen analog coding sequence into a pRMG5 construct and then further subcloning into the expression vector pLGD43.
- trypsinogen analog gene sequences Two different trypsinogen analog gene sequences were cloned into pLGD43. The two gene sequences encoded a trypsinogen analog with either four or six amino acids in the leader sequence wherein the lysine is replaced with aspartate (Val(Asp) 5 -trypsin or Val(Asp)3-trypsin ).
- the trypsinogen analog having the four amino acid leader sequence was cloned using two oligonucleotides, 5'-TC GAG GGT AAC CTT TAT TTC CCT TCT TTT TCT CTT TAG CTC GGC TTA TTC CAG GGG TGT GTT TCG TCG AGT CGA CGA CGA T-3' and 5'-ATC GTC GTC GAC TCG ACG AAA CAC ACC CCT GGA ATA AGC CGA GCT AAA GAG AAA AAG AAG GGA AAT AAA GGT TAC CC-3', encoding the HSA signal sequence and the four amino acid leader sequence.
- the oligos were phosphorylated, annealed and ligated into the Xhol and EcoRV sites of the pRMG5- ⁇ F( ⁇ A) 2 VD5 construct described above to create pRMG5HSAVD3.
- the resulting pRMG5 vector was then used for transferring the DNA sequence encoding the HSA signal sequence fused to the four amino acid leader sequence to pLGD43 as a BstElllBam ⁇ l fragment to create pLGD43HSAVD3.
- the trypsinogen analog having the six amino acid leader sequence was cloned by taking the trypsinogen analog gene cassette from pRMG5- ⁇ F( ⁇ A) 2 VD5 as a SaWBamHl fragment into the vector pRMG5HSAVD3 also digested with Sail and BamHl.
- the resulting vector was named pRMG5HSAVD5.
- This modified trypsinogen was then subcloned from pRMG5HS AVD5 into pLGD43 as described above for the modified trypsinogen containing the four amino acid leader sequence.
- the resulting vector was named pLGD43HSAVD5.
- Pichia pastoris GS115 and SMDl 163 protease minus strains were transfected using the spheroplast method or the electroporation method.
- the expression vectors described in Example 1 were linearized with BgHl for pPIC9 derivatives and N ⁇ tl for pLGD43 derivatives to facilitate homologous recombination of an expression cassette on the Pichia chromosome.
- This cassette consisted of a promoter (AOXl or GAPDH) controlling the expression of a trypsinogen analog and the Yeast His4 gene which was used as a selectable marker.
- Yeast strains used for transformation have a mutated HIS4 gene and are unable to grow in medium lacking histidine.
- Transfected cultures were plated on minimal medium and only those cells which had integrated the HIS4 gene in their chromosome were able to grow on minimal medium lacking histidine (HIS+ clones).
- Clones having integrated the cassette in the AOXl locus were then isolated. Homologous recombination directed to the AOXl locus leads to the deletion of the AOXl gene. Strains carrying a deletion of the AOXl gene are still able to grow on methanol using AOX2 alcohol oxydase but the growth is much slower compared to cells with an intact AOXl gene. Therefore, HIS+ transformants were screened for integration in the AOXl locus by plating them on minimal medium containing methanol as the sole carbon source. Slow growing clones having integrated the expression cassette in the AOXl locus were isolated and designated mut s (methanol utilization slow).
- HIS+/mut cells which were originally transfected with the expression vectors containing trypsinogen analogs driven by the AOXl promoter, were first grown in glycerol to generate biomass and then transferred to medium containing methanol for induction. Clones to be screened were stored as 1 ml glycerol stocks at -80°C.
- BMGY lOOmM phosphate buffer pH 6.0, 1.34% bacto yeast nitrogen base (YNB), 10% yeast extract, 20% bactopeptone, 1% glycerol.
- BMMY lOOmM phosphate buffer pH 6.0, 1.34% YNB, 10% yeast extract, 20% bactopeptone, 0.5% methanol
- 125 mis of pure methanol was added to keep the level of methanol constant in the culture medium. The cultures were centrifuged and the supernatant was isolated to determine the concentration of trypsinogen analog.
- the GAPDH promoter is induced when glucose becomes limited in the culture medium.
- the induction of expression of trypsinogen analog from the GAPDH promoter was carried out s essentially as described for the AOXl promoter.
- Subconfluent cultures of HIS+/mut cells which were originally transfected with expression vectors containing a trypsinogen analog driven by the GAPDH promoter, were grown in BMGlcY (lOOmM phosphate buffer pH6, 1.34% YNB, 10% yeast extract, 20% bactopeptone, 5% glucose). Cells were harvested and transferred to BMGY medium depleted in glucose (lOOmM phosphate buffer pH6, 1.34% YNB, 10% yeast extract, 20% bactopeptone). The supernatant was harvested 48 hours after induction.
- Example 4 Selection and characterization of clones expressing trypsinogen analogs: The trypsinogen present in the supernatant of Pichia pastoris was first concentrated by ammonium sulfate precipitation (70%). The precipitate was resuspended in 20 mis of 20mM sodium acetate pH 5 and dialyzed overnight against the same buffer at 4°C. The dialyzed solution was then loaded on CM-Sepharose CL6B (Pharmacia, Upsala, Sweden) equilibrated with 20mM acetate buffer pH 5. Trypsinogen analog was eluted with a gradient of NaCl (0- 50mM). Fractions were analyzed by SDS-PAGE and fractions containing the trypsinogen analogs were pooled and concentrated.
- the purified trypsinogen was then activated with 500 U dDAP per gram of trypsinogen analog in 50 mM acetate buffer pH 3.0 and the resulting trypsin activity was determined using the Tosyl-Arg methyl esterase (TAME) assay as described in B.C.W. Hummel, 37 Can. J. Biochem. Physiol., 1393 (1959).
- TAME Tosyl-Arg methyl esterase
- FIG. 3 summarizes the production of ValAsp5 trypsinogen in the two expression systems described in the preceding Examples.
- N-terminal sequencing was carried out on purified trypsinogen analog expressed from two different constructs.
- the 25kDa and 30 kDa bands observed on SDS gels were sequenced which confirmed that the two proteins were trypsinogen.
- Both the 30kDa and the 25 kDa proteins were found with an N-terminus corresponding to modified trypsinogen.
- the trypsinogen produced by Pichia pastoris is secreted into the culture medium. Both the alpha factor and the HSA signal sequence are correctly processed during secretion, but as mentioned above the GluAlaGluAla sequence inserted between the signal sequence and the trypsinogen is only partially removed.
- Trypsinogen analog was treated with dDAP and trypsin activity followed with time. Trypsinogen purified from Pichia culture supernatant was activatable into trypsin with dDAP. In the absence of dDAP, no trypsin activity was detected. Following incubation with dDAP, the trypsin activity increases and reaches a plateau once all activatable trypsinogen has been digested with dDAP. Animal sourced trypsinogen purchased from Sigma Chemical Company was also activated into trypsin by dDAP but the trypsin activity started to decrease after a few hours. The Sigma trypsin produced by dDAP activation at pH 3.0 was much less stable then the trypsin produced from trypsinogen analog expressed in Pichia.
- N-terminal sequencing was done on the trypsin produced following dDAP digestion .and the N-terminus was found to be identical to wild type bovine trypsin.
- the dDAP removes only aminoacids up to He 11 (SEQ ID NO:2) and does not digest further.
- This example sets out methods useful in assessing the quality of the inventive products, especially the essentially chymotrypsin-free products.
- the purity of r-trypsin is measured by SDS-Page under non-reducing conditions, followed by densitometric analysis of the bands.
- the potential impurities that can be measured are small fragments of trypsin or yeast polypeptides.
- the method is designed to measure contamination of the r-trypsin by yeast proteins from the fermentation. The method also detects trypsin, two chain trypsin, and two other proteolysis products of trypsin. These variants of trypsin are counted as product in the calculation of purity. All bands included as trypsin product have been identified as bovine trypsin sequences as measured by direct sequence analysis of bands from control sample of bovine trypsin eluted from gels. Bands found at all other molecular weights are considered impurities.
- the activity of trypsin can be measured by following the degradation of a synthetic substrate (TAME or Tosyl-Arg-Methyl-Ester) as measured by a change in absorbance at 247 nm over time. This method is essentially as described by Hummel, Can. J. Biochem. Physiol. 37: 1393 (1959).
- the specific activity (expressed in Units/mg) is calculated as the ratio between the activity (expressed in Units per ml) and the protein concentration (expressed in mg/ml).
- high quality trypsin will have a specific activity of >190U/mg.
- the specific activity should exceed 210U/mg, with superior preparations exceeding 220 U/mg.
- the specificity of a lot of r-trypsin is determined by performing a complete tryptic digest of a reference standard of glandular glucagon at room temperature using a ratio of 1 mg of trypsin per 100 mg of substrate over a period of 2 hours, as described below. This exposure-ratio combination is equivalent to the reaction conditions in most processes using trypsin. The reaction is monitored by reversed-phase HPLC, as described below. The results are reported as the ratio of the sum of the area for the tryptic fragments obtained with a sample versus that obtained with a standard (% specificity). This assay was designed to measure any degradation of the substrate resulting from contaminating proteases (particularly chymotrypsin) which could have co-purified with r-trypsin throughout the purification process.
- Glucagon contains several chymotryptic sites and the assay can detect very low levels of chymotryptic contamination of trypsin (>0.01% by comparing the area of chymotryptic peaks to the area of tryptic peaks).
- Example 6 HPLC Method for Detecting Trypsin versus Chymotrypsin This assay is useful in measuring the purity of trypsin and simultaneously measuring chymotryptic activity.
- Glucagon Substrate Preparation One vial of glucagon reference standard is needed to assay two trypsin samples in triplicate. To each vial of glucagon reference standard, add 0.5 mL of 0.001 M HC1. Mix gently, and transfer the solution to a 15 mL polypropylene tube. Add 5 mL of the 50 mM borate buffer. Add 28 ⁇ L of the 1 M CaCl 2 stock solution and mix. Measure the pH and adjust to 8.0 +/- 0.1 with IN NaOH if necessary. Hold the solution on ice until needed. The same substrate solution must be used for analysis of both the bovine trypsin (bTrp) control and the recombinant trypsin (rTrp) samples.
- bTrp bovine trypsin
- rTrp recombinant trypsin
- Glucagon Standard Preparation Aliquot 250 ⁇ L of the glucagon solution prepared in step a) into a 1.5 mL microfuge tube. Add 750 ⁇ L of the 50 mM borate buffer and mix. Add 50 ⁇ L of the 5 N HC1 and mix. Hold on ice until needed.
- bTrp Standard Preparation Weigh out approximately 1 mg of the Sigma bovine trypsin. Dissolve in 1 mL of 0.05 M HOAc. Determine the concentration of the bTrp solution by measuring the A280 on the Spectrophotometer. See Data Analysis section for calculations. Dilute the solution to 0.5 mg/mL with 0.05 M HOAc. Hold on ice until needed.
- rTrp Sample Preparation Determine the concentration of the rT ⁇ * s3rrr le by measuring the A280 on the Spectrophotometer. See Data Analysis for calculations. Dilute the solution to 0.5 mg/mL with 0.05 M HOAc. Hold on ice until needed.
- Enzyme reaction For each sample, transfer a 1 mL aliquot of the glucagon reference standard substrate solution into a 1.5 mL eppendorf tube. Add 10 ⁇ L of the 0.5 mg/mL bTrp standard solution or rTrp sample solution and vortex. Place each tube in a 25°C water bath and incubate for 2 hours. After 2 hours, quench the reaction by adding 50 ⁇ L of 5 N HCL Samples must be held between 4 and 9°C prior to analysis or precipitation will occur. Samples should be analyzed within 12 hours after quenching. The bTrp standard must be analyzed using the same glucagon substrate solution and in the same HPLC sequence as the rTrp samples.
- Needle Wash 1000 ⁇ L after each injection. See Materials #8, above, for wash solution makeup.
- a typical HPLC run of trypsin-digested material yields 4 tryptic peaks at retention time of about 6.1 (3), 9.1 (2), 17.4 (1) and 18.7 (4) minutes (parentheticals indicate the ranking of the peaks in order of area, largest to smallest).
- the same protocol used with chymotrypsin yielded 5 peaks having retention time of about 8.0 (3), 8.1 (2), 8.7 (5) , 13.5 (1) and 13.6 (4) minutes (parentheticals indicate the ranking of the peaks in order of area, largest to smallest).
- all chymotryptic and tryptic peaks resulting from glucagon digestion are clearly resolvable.
- Standard trypsin samples were spiked with 1%, 0.1% and 0.01% (by weight) of chymotrypsin (Sigma) and the reaction resolved by the same HPLC method. Peak resolution was obtained at all spiking levels, demonstrating that the detection limit of the assay is well below 0.01% by weight.
- the sensitivity may be enhanced even further.
- the inherent limit on sensitivity is interference with the chymotryptic peaks by the tryptic peaks. Sensitivity may be increased, it is believed, to levels of less than 0.005%, 0.001% and even 0.0005%, merely by increasing the amount of material injected into the HPLC system.
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WO2001019970A2 (en) * | 1999-09-15 | 2001-03-22 | Eli Lilly And Company | Chymotrypsin-free trypsin |
CN1545553B (zh) * | 2001-02-01 | 2011-09-21 | 弗·哈夫曼-拉罗切有限公司 | 生产重组胰蛋白酶的方法 |
DE60328802D1 (de) | 2002-08-30 | 2009-09-24 | Novozymes Inc | Verfahren zur herstellung von säugetier-trypsinen |
CN104312933B (zh) * | 2014-10-17 | 2017-03-29 | 江南大学 | 一种优化信号肽提高胰蛋白酶胞外分泌表达的方法 |
CN108463553A (zh) | 2015-11-25 | 2018-08-28 | 莱顿教学医院 | 重组丝氨酸蛋白酶 |
CN116445462A (zh) * | 2023-04-20 | 2023-07-18 | 西安麦博泰克生物科技有限公司 | 一种重组猪胃蛋白酶的纯化制备方法及重组猪胃蛋白酶 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0597681A1 (de) * | 1992-11-13 | 1994-05-18 | Eli Lilly And Company | Expressionsvektore für Rinder-Trypsine und Trypsinogene und damit transformierte Wirtzellen |
Family Cites Families (4)
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US5763215A (en) * | 1984-08-16 | 1998-06-09 | Bio-Technology General Corporation | Method of removing N-terminal amino acid residues from eucaryotic polypeptide analogs and polypeptides produced thereby |
TW257792B (de) * | 1992-10-01 | 1995-09-21 | Lilly Co Eli | |
US5773248A (en) * | 1995-11-13 | 1998-06-30 | Uab Research Foundation | Nucleic acid encoding a human α3(IX) collagen protein and method of producing the protein recombinantly |
JPH09294583A (ja) * | 1996-03-08 | 1997-11-18 | Ajinomoto Co Inc | アミノペプチダーゼgx及びそれを用いるタンパク質の加水分解方法 |
-
1999
- 1999-09-15 CA CA002343966A patent/CA2343966A1/en not_active Abandoned
- 1999-09-15 EP EP99951445A patent/EP1141263A4/de not_active Withdrawn
- 1999-09-15 WO PCT/US1999/021047 patent/WO2000017332A1/en not_active Application Discontinuation
- 1999-09-15 AU AU63884/99A patent/AU6388499A/en not_active Abandoned
- 1999-09-20 AR ARP990104729A patent/AR021818A1/es unknown
- 1999-09-20 PE PE1999000952A patent/PE20001068A1/es not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0597681A1 (de) * | 1992-11-13 | 1994-05-18 | Eli Lilly And Company | Expressionsvektore für Rinder-Trypsine und Trypsinogene und damit transformierte Wirtzellen |
Non-Patent Citations (1)
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See also references of WO0017332A1 * |
Also Published As
Publication number | Publication date |
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AR021818A1 (es) | 2002-08-07 |
PE20001068A1 (es) | 2000-10-18 |
WO2000017332A1 (en) | 2000-03-30 |
EP1141263A1 (de) | 2001-10-10 |
AU6388499A (en) | 2000-04-10 |
CA2343966A1 (en) | 2000-03-30 |
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