WO1997000316A1 - A process for producing trypsin (trypsinogen) - Google Patents
A process for producing trypsin (trypsinogen) Download PDFInfo
- Publication number
- WO1997000316A1 WO1997000316A1 PCT/DK1996/000253 DK9600253W WO9700316A1 WO 1997000316 A1 WO1997000316 A1 WO 1997000316A1 DK 9600253 W DK9600253 W DK 9600253W WO 9700316 A1 WO9700316 A1 WO 9700316A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- trypsinogen
- host
- trypsin
- derivative
- dna sequence
- Prior art date
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Classifications
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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)
-
- 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
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
Definitions
- the present invention relates to a process for the production of trypsins in filamentous fungi and to DNA sequences to be used in such processes.
- This method is generally used for the expression and production of proteins originating from other microbial sources, but mammalian proteins have also been produced in such systems.
- the present invention relates to a process for the production of trypsins (trypsinogens) or derivatives thereof in filamentous fungi, the process comprising
- the term "derivative" is intended to indicate a polypeptide which is derived from the native trypsin or signal peptide (as the case may be) by suitably modifying the DNA sequence coding for the native trypsin/signal peptide, resulting in the addition of one or more amino acid at either or both the C- or N-terminal end, substitution of one or more amino acids at one or a number of different sites in the native amino acid sequence, deletion of one or more amino acids at either or both ends of the native amino acid sequence or at one or more sites within the native sequence, or insertion of one or more amino acids at one or more sites in the native amino acid sequence.
- modifications of the DNA sequence may be done by methods well known in the art.
- filamentous fungus is intended to include the groups Phycomycetes, Zygomycetes, Ascomycetes, Basidiomycetes and fungi imperfecti, including Hyphomycetes such as the genera Aspergillus, Penicillium, Trichoderma, Fusarium and Humicola.
- the presence of the signal sequence serves to direct the expressed trypsinogen or derivative thereof effectively into the secretory pathway of the host cell so that trypsinogen or trypsin may readily be isolated from the culture medium (at least some of the product recovered will be mature trypsin as the trypsinogen secreted from the cells is either subjected to automaturation or maturation by proteases produced by the host cell) .
- the signal sequence does not seem to be critical, and a number have been tested, such as the TAKA- amylase (ref. EP 0 238 023), the PTRYP-trypsin, and the human HTRYPI-trypsin and HTRYPII signal sequences (Okaya a et al., Methods in Enzymology 154, 3-28 (1987), Emi et al., Gene 41, 305-310, (1986)).
- the trypsin (trypsinogen) to be produced by the process of the invention is trypsin of any origin, especially mammalian trypsin, such as porcine, bovine, and human trypsin.
- the invention furthermore comprises certain DNA sequences coding for porcine trypsin (trypsinogen) and alleles thereof capable of expressing trypsins having retained their biological activity.
- the invention relates to vectors comprising said DNA sequence and hosts transformed therewith.
- Fig.l shows the steps involved in the construction of pHW470
- Fig. 2 shows the steps involved in the construction of pHW473.
- Fig. 3 shows the steps involved in the construction of pHW874,
- the present invention in its first aspect relates to a process for the production of trypsins (trypsinogens) or derivatives thereof in filamentous fungi, the process compris ⁇ ing
- the vector may further comprise DNA sequences encoding functions facilitating gene expression, typically a promoter, transcription initiation sites, and transcription termination and polyadenylation functions.
- the promoter which may be preceded by upstream activating sequences and enhancer sequences as known in the art may be any DNA sequence exhibiting a strong transcriptional activity in Aspergillus sp. , such as A ⁇ . oryzae and A ⁇ . niger, and may be derived from a gene encoding an extracellular or intracellular protein such as an amylase, a glucoamylase, a protease, a lipase, a cellulase or a glycolytic enzyme.
- promoters examples include those derived from the gene encoding A ⁇ . orvzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.;, niger neutral ⁇ -amylase, A ⁇ _ niger acid stable ⁇ - amylase, A ⁇ niger glucoamylase, Rhizomucor miehei lipase, or A. oryzae alkaline protease.
- promoters from genes encoding glycolytic enzymes are the A ⁇ _ oryzae triose phosphate isomerase, ADH and PGK promoters.
- the filamentous fungus used as the host organism is preferably selected from an Aspergillus sp. such as A ⁇ niger A. awamori or A. oryzae.
- Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
- the techniques used to transform the host organism may suitably be adapted from the methods of transforming A_ j _ nidulans described in, for instance, Yelton et al., Proc. Natl. Acad. Sci. USA 81, 1984, pp. 1470-1474, or EP 0 215 594, from the methods of transforming A ⁇ niger described in, for instance Buxton et al., Gene 37, 1985, pp. 207-215 or US 4,885,249, or from the methods of transforming A ⁇ oryzae described in EP 238023.
- a ⁇ . orvzae or A In the process of the present invention, A ⁇ . orvzae or A.
- niger may be transformed with a vector system comprising a DNA sequence coding for a selection marker which is capable of being incorporated in the genome of the host organism on transformation, but which is either not expressed by the host before transformation or not expressed in sufficient amounts to permit growth under selective conditions. Transformants can then be selected and isolated from non-transformants on the basis of the incorporated selection marker.
- Suitable selection markers may be derived from the A.-, nidulans or A_i. niger argB gene, the A ⁇ _ nidulans trpC gene, the A. nidulans amdS gene, the Neurospora crassa pyr4 or DHFR genes, or the Ai. niger or A ⁇ orvzae niaD gene.
- Preferred selection markers for use in the present invention are derived from the A ⁇ . nidulans or A ⁇ niger amdS or argB genes. If argB is chosen as the selection marker, an ArgB " mutant strain (which does not express the ArgB gene) must be used as the host organism. On the other hand, the amdS gene may be used as the selection marker in wild-type A ⁇ oryzae or A. niger strains which do not express this gene in sufficient amounts to permit growth under selective conditions.
- the signal sequence may be chosen from signal sequences derived from the trypsinogen gene itself, or from a gene encoding e.g. A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase,
- A. niger neutral ⁇ -amylase A ⁇ . niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, or A ⁇ oryzae alkaline protease.
- genes encoding glycolytic enzymes are the A ⁇ _ orvzae triose phosphate isomerase, ADH and PGK. Combinations and/or variants of such signal sequences may also be used.
- the gene coding for trypsinogen fused to the signal sequence as well as to promoter and terminator sequences may be inserted in a vector containing the selection marker, or it may be inserted in a separate vector for introduction into the host cell.
- the vector or vectors may be linear or closed circular molecules.
- the medium used to culture the transformed host cells may be any conventional medium suitable for growing filamentous fungi.
- the transformants are usually stable and may be cultured in the absence of selection pressure. However, if the transformants are found to be unstable, the selection marker introduced into the cells may be used for selection.
- the trypsinogen or trypsin produced by the host cells may conveniently be recovered from the culture medium by well-known procedures including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
- the invention furthermore comprises certain DNA sequences coding for porcine trypsin (trypsinogen) and alleles thereof capable of expressing trypsins having retained their biological activity.
- the invention relates in a further aspect to vectors comprising said DNA sequences.
- the invention also encompasses hosts transformed with such vectors.
- the hosts may be of animal or microbial origin, such as mammalian cell lines, bacteria, yeasts or fungi, especially filamentous fungi.
- the invention relates to a method of recombinantly producing porcine trypsin, the process comprising
- NOR 948 5 1 GCCCCCAACGATCTTGTCATCATCATC 3' SEQ ID NO: 3 NOR 949 : 5' GTTCAGAGTCTTCCTGTCGTATTGGGG 3' SEQ ID NO: 4
- NOR 948 is common to TRYI and TRYII
- NOR 949 is specific for TRYII.
- Full length clones were isolated having sequences in accordance with the ones published by Emi et al., Gene 41 , 305- 310 (1986) .
- the plasmids were designated pHW468 for TRYI and pHW469 for TRYII.
- Example 2 The plasmids were designated pHW468 for TRYI and pHW469 for TRYII.
- mRNA was purified from porcine pancreas using standard methods (Maniatis 1982) .
- cDNA was prepared from the mRNA, purified and inserted into ⁇ gtll using the cDNA cloning system- ⁇ gtll from Amersham, UK. Preparation of phage, plating cells, infection with ⁇ gtll, amplification and screening was performed according to the manufacturers introductions and standard techniques (Maniatis 1982) .
- Positive plaques were isolated and amplified.
- the isolated ⁇ gtll DNA was subjected to digestion with EcoRI and the inserted cDNA was cloned into EcoRI cleaved pBluescript SK (Stratagene) using ampicillin selection of E ⁇ . coli JM101 transformants.
- the selected plasmid was shown by DNA sequencing analysis (Sequenase, U.S. Biochemical Corp.) to contain a cDNA sequence compatible with the known porcine trypsin amino acid sequence (Hermodson et al., Biochemistry . 12 ./ 3146-3153 (1973)).
- NOR 971 5' GATCCACCATGAATCCACTCCTGATCCTTACCTTTGTGGCAG 3' NOR 972 : 3' GTGGTACTTAGGTGAGGACTAGGAATGGAAACACCGTC 5' SEQ ID NO: 5
- the common linker covers the first 11 amino acids of the signal sequence of TRYI, differing only in position 3 from TRYII, which has a leucine instead of proline in its native sequence. The remaining part of the sequence is native to both species.
- the trypsinogen expression vectors pHW470 and pHW473 were transformed into A ⁇ oryzae IFO 4177, or a protease deficient derivative thereof, A1560-T40, using the procedure described in EP 238023. Selection on acetamide was performed by co- transformation with pToC 186 as described in WO 93/00426.
- a vector for expression of porcine trypsinogen in Aspergillus was constructed as outlined in Fig. 3. To connect the first 18 amino acids of the TAKA amylase signal to the last 4 amino acids of the porcine trypsin signal, we used a Banl-EcoRl linker :
- This fusion also has a part of the TAKA amylase promoter and the N-terminal end of the trypsin gene.
- the C-terminal region of the trypsin gene is joined to this in Sub2, keeping track of the orientations.
- the final expression vector, pHW874, has TAKA amylase promoter and AMG terminator as functional elements. These elements were derived from pHD414, which is described in EP 0 505 311.
- the porcine trypsin expression vector pHW874 was transformed into A ⁇ _ oryzae as described in Example 3. Transformants were grown in YPD medium and analysed by SDS-PAGE-Western and by cleavage of L-BAPNA, as described in Example 3. In this case distinct bands of the expected size for porcine trypsinogen and mature trypsin were seen on Western blots, corresponding to activity measurements with L-BAPNA.
- AAA AAC AAG CCT GGG GTC TAC ACC AAG GTC TGC AAC TAT GTG AAC TGG 720 Lys Asn Lys Pro Gly Val Tyr Thr Lys Val Cys Asn Tyr Val Asn Trp 225 230 235
- MOLECULE TYPE DNA probe
- HYPOTHETICAL YES
- ANTI-SENSE NO
- MOLECULE TYPE DNA probe
- HYPOTHETICAL YES
- ANTI-SENSE NO GTTCAGAGTC TTCCTGTCGT ATTGGGG
- MOLECULE TYPE DNA linker
- HYPOTHETICAL YES
- ANTI-SENSE NO GATCCACCAT GAATCCACTC CTGATCCTTA CCTTTGTGGC AG
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Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8533583A JPH11507207A (en) | 1995-06-16 | 1996-06-10 | Method for producing trypsin (trypsinogen) |
EP96918619A EP0833898A1 (en) | 1995-06-16 | 1996-06-10 | A process for producing trypsin (trypsinogen) |
AU61217/96A AU6121796A (en) | 1995-06-16 | 1996-06-10 | A process for producing trypsin (trypsinogen) |
US08/956,267 US5945328A (en) | 1995-06-16 | 1997-10-22 | Process for producing trypsin (trypsinogen) |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DK69395 | 1995-06-16 | ||
DK0693/95 | 1995-06-16 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08/956,267 Continuation US5945328A (en) | 1995-06-16 | 1997-10-22 | Process for producing trypsin (trypsinogen) |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997000316A1 true WO1997000316A1 (en) | 1997-01-03 |
Family
ID=8096464
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/DK1996/000253 WO1997000316A1 (en) | 1995-06-16 | 1996-06-10 | A process for producing trypsin (trypsinogen) |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0833898A1 (en) |
JP (1) | JPH11507207A (en) |
AU (1) | AU6121796A (en) |
WO (1) | WO1997000316A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999010503A1 (en) * | 1997-08-22 | 1999-03-04 | Roche Diagnostics Gmbh | Zymogenic protease precursors that can be autocatalytically activated and their use |
WO2001055429A2 (en) * | 2000-01-24 | 2001-08-02 | Polymun Scientific Immunbiologische Forschung Gmbh | Method for the manufacture of recombinant trypsin |
US6420156B2 (en) * | 1997-08-22 | 2002-07-16 | Nestec S.A. | Purified proteolytic enzyme and method of purification |
WO2002061064A2 (en) * | 2001-02-01 | 2002-08-08 | Roche Diagnostics Gmbh | Method for producing recombinant trypsin |
WO2004020612A1 (en) | 2002-08-30 | 2004-03-11 | Novozymes Biotech, Inc. | Methods for producing mammalian trypsins |
US7351549B2 (en) * | 2000-01-24 | 2008-04-01 | Polymun Scientific Immunbiologische Forschung Gmbh | Method for the manufacture of recombinant trypsin |
WO2011030347A1 (en) | 2009-09-10 | 2011-03-17 | Biocon Limited | Novel prolipase-bovine trypsinogen fusion proteins |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0597681A1 (en) * | 1992-11-13 | 1994-05-18 | Eli Lilly And Company | Expression vectors for the bovine trypsin and trypsinogen and host cells transformed therewith |
WO1994025583A1 (en) * | 1993-05-05 | 1994-11-10 | Novo Nordisk A/S | A recombinant trypsin-like protease |
WO1995015391A2 (en) * | 1993-12-01 | 1995-06-08 | Novo Nordisk Biotech, Inc. | Aspergillus expression system |
-
1996
- 1996-06-10 EP EP96918619A patent/EP0833898A1/en not_active Ceased
- 1996-06-10 WO PCT/DK1996/000253 patent/WO1997000316A1/en not_active Application Discontinuation
- 1996-06-10 AU AU61217/96A patent/AU6121796A/en not_active Abandoned
- 1996-06-10 JP JP8533583A patent/JPH11507207A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0597681A1 (en) * | 1992-11-13 | 1994-05-18 | Eli Lilly And Company | Expression vectors for the bovine trypsin and trypsinogen and host cells transformed therewith |
WO1994025583A1 (en) * | 1993-05-05 | 1994-11-10 | Novo Nordisk A/S | A recombinant trypsin-like protease |
WO1995015391A2 (en) * | 1993-12-01 | 1995-06-08 | Novo Nordisk Biotech, Inc. | Aspergillus expression system |
Non-Patent Citations (1)
Title |
---|
DATABASE WPI 1 January 1900 Derwent World Patents Index; AN 1988-224890, XP002978867, "Human Spleen Trypsin - Used to Treat Lesions or Trauma, without Hypersensitive Allergic Side Effects" * |
Cited By (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6420156B2 (en) * | 1997-08-22 | 2002-07-16 | Nestec S.A. | Purified proteolytic enzyme and method of purification |
WO1999010503A1 (en) * | 1997-08-22 | 1999-03-04 | Roche Diagnostics Gmbh | Zymogenic protease precursors that can be autocatalytically activated and their use |
US7351549B2 (en) * | 2000-01-24 | 2008-04-01 | Polymun Scientific Immunbiologische Forschung Gmbh | Method for the manufacture of recombinant trypsin |
WO2001055429A2 (en) * | 2000-01-24 | 2001-08-02 | Polymun Scientific Immunbiologische Forschung Gmbh | Method for the manufacture of recombinant trypsin |
WO2001055429A3 (en) * | 2000-01-24 | 2002-05-16 | Polymun Scient Immunbio Forsch | Method for the manufacture of recombinant trypsin |
US7666629B2 (en) | 2001-02-01 | 2010-02-23 | Roche Diagnostics Operations, Inc. | Method for producing recombinant trypsin |
US7276605B2 (en) | 2001-02-01 | 2007-10-02 | Roche Diagnostics Operations, Inc. | Method for producing recombinant trypsin |
WO2002061064A3 (en) * | 2001-02-01 | 2003-12-24 | Roche Diagnostics Gmbh | Method for producing recombinant trypsin |
WO2002061064A2 (en) * | 2001-02-01 | 2002-08-08 | Roche Diagnostics Gmbh | Method for producing recombinant trypsin |
CZ303658B6 (en) * | 2001-02-01 | 2013-02-06 | F. Hoffmann-La Roche Ag | Production process of recombinant trypsin |
WO2004020612A1 (en) | 2002-08-30 | 2004-03-11 | Novozymes Biotech, Inc. | Methods for producing mammalian trypsins |
WO2011030347A1 (en) | 2009-09-10 | 2011-03-17 | Biocon Limited | Novel prolipase-bovine trypsinogen fusion proteins |
CN102482675A (en) * | 2009-09-10 | 2012-05-30 | 拜康有限公司 | Novel prolipase-bovine trypsinogen fusion proteins |
KR101410845B1 (en) * | 2009-09-10 | 2014-06-23 | 바이오콘 리미티드 | Novel prolipase-bovine trypsinogen fusion proteins |
US8975041B2 (en) | 2009-09-10 | 2015-03-10 | Biocon Limited | Fusion proteins and method of expression thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0833898A1 (en) | 1998-04-08 |
AU6121796A (en) | 1997-01-15 |
JPH11507207A (en) | 1999-06-29 |
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