CA2437342C - Method for producing recombinant trypsin - Google Patents
Method for producing recombinant trypsin Download PDFInfo
- Publication number
- CA2437342C CA2437342C CA2437342A CA2437342A CA2437342C CA 2437342 C CA2437342 C CA 2437342C CA 2437342 A CA2437342 A CA 2437342A CA 2437342 A CA2437342 A CA 2437342A CA 2437342 C CA2437342 C CA 2437342C
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- trypsinogen
- trypsin
- host cell
- expression
- amino acid
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- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000000813 peptide hormone Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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)
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP01102342.1 | 2001-02-01 | ||
| EP01102342 | 2001-02-01 | ||
| PCT/EP2002/001072 WO2002061064A2 (de) | 2001-02-01 | 2002-02-01 | Verfahren zur herstellung von rekombinantem trypsin |
Publications (2)
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|---|---|
| CA2437342A1 CA2437342A1 (en) | 2002-08-08 |
| CA2437342C true CA2437342C (en) | 2013-08-06 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2437342A Expired - Fee Related CA2437342C (en) | 2001-02-01 | 2002-02-01 | Method for producing recombinant trypsin |
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|---|---|
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| EP (1) | EP1399568B1 (enExample) |
| JP (1) | JP4159878B2 (enExample) |
| CN (1) | CN1545553B (enExample) |
| AT (1) | ATE310092T1 (enExample) |
| CA (1) | CA2437342C (enExample) |
| CZ (1) | CZ303658B6 (enExample) |
| DE (1) | DE50204952D1 (enExample) |
| DK (1) | DK1399568T3 (enExample) |
| WO (1) | WO2002061064A2 (enExample) |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002061064A2 (de) * | 2001-02-01 | 2002-08-08 | Roche Diagnostics Gmbh | Verfahren zur herstellung von rekombinantem trypsin |
| DK1546302T3 (da) | 2002-08-30 | 2009-12-14 | Novozymes Inc | Fremgangsmåder til fremstilling af pattedyrs-trypsiner |
| ES2370657T3 (es) * | 2005-09-14 | 2011-12-21 | Sanofi-Aventis Deutschland Gmbh | Escisión de precursores de insulinas mediante una variante de tripsina. |
| AU2009336710A1 (en) * | 2009-01-06 | 2011-06-30 | Nestec S.A. | Processing of macronutrients |
| CN101967469B (zh) * | 2009-07-28 | 2012-11-14 | 上海雅心生物技术有限公司 | 一种高稳定性的重组胰蛋白酶的生产及应用 |
| CN102482675B (zh) * | 2009-09-10 | 2015-04-29 | 拜康有限公司 | 新型脂酶原-牛胰蛋白酶原融合蛋白 |
| WO2012104099A1 (en) | 2011-02-04 | 2012-08-09 | Glucometrix Ag | Process for the production of recombinant trypsin |
| CN103805584B (zh) * | 2012-11-12 | 2016-08-24 | 宜昌东阳光长江药业股份有限公司 | 一种重组胰蛋白酶纯化方法 |
| CN103405756B (zh) * | 2013-07-10 | 2014-12-24 | 浙江众益制药股份有限公司 | 药物组合物复方消化酶胶囊(ii)及其制备方法 |
| MA39835A (fr) | 2014-04-17 | 2017-02-22 | Biogen Ma Inc | Compositions et procédés de modulation de l'épissage du smn2 chez un patient |
| CN104046605A (zh) * | 2014-05-29 | 2014-09-17 | 中国科学院广州能源研究所 | 一种嗜温耐乙醇β-葡萄糖苷酶及其编码基因和应用 |
| US10436802B2 (en) | 2014-09-12 | 2019-10-08 | Biogen Ma Inc. | Methods for treating spinal muscular atrophy |
| JP6686361B2 (ja) * | 2014-11-26 | 2020-04-22 | 東ソー株式会社 | 細胞回収のための標本作製方法 |
| WO2016164896A2 (en) | 2015-04-10 | 2016-10-13 | Ionis Pharmaceuticals, Inc. | Modulation of smn expression |
| WO2017218884A1 (en) | 2016-06-16 | 2017-12-21 | Ionis Pharmaceuticals, Inc. | Combinations for the modulation of smn expression |
| CN106350497B (zh) * | 2016-11-24 | 2019-07-16 | 天津大学 | 一种胰蛋白酶及其制备方法与应用 |
| CN110511916B (zh) * | 2019-08-06 | 2021-06-01 | 杭州浦泰生物科技有限公司 | 一种重组胰蛋白酶的生产工艺 |
| AR121446A1 (es) | 2020-02-28 | 2022-06-08 | Ionis Pharmaceuticals Inc | Compuestos y métodos para modular smn2 |
| CN115216463B (zh) * | 2022-06-15 | 2023-08-15 | 武汉瀚海新酶生物科技有限公司 | 具有稳定性的重组胰蛋白酶及其制备方法和应用 |
| CN120225684A (zh) | 2022-11-23 | 2025-06-27 | 豪夫迈·罗氏有限公司 | 用于增加重组蛋白表达的方法 |
Family Cites Families (39)
| Publication number | Priority date | Publication date | Assignee | Title |
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| DE249282C (enExample) | ||||
| US4851341A (en) * | 1986-12-19 | 1989-07-25 | Immunex Corporation | Immunoaffinity purification system |
| US5011912A (en) * | 1986-12-19 | 1991-04-30 | Immunex Corporation | Hybridoma and monoclonal antibody for use in an immunoaffinity purification system |
| US7247453B1 (en) * | 1988-12-30 | 2007-07-24 | Oklahoma Medical Research Foundation | Calcium binding recombinant antibody against protein C |
| US5298599A (en) * | 1988-12-30 | 1994-03-29 | Oklahoma Medical Research Foundation | Expression and purification of recombinant soluble tissue factor |
| US6433142B1 (en) * | 1989-08-08 | 2002-08-13 | Genetics Institute, Llc | Megakaryocyte stimulating factors |
| US5646016A (en) * | 1991-02-06 | 1997-07-08 | Genetics Institute, Inc. | Peptide and protein fusions to thioredoxin, thioredoxin-like molecules, and modified thioredoxin-like molecules |
| CA2102673A1 (en) | 1992-11-13 | 1994-05-14 | Michael G. Greaney | Expression vectors for bovine trypsin and trypsinogen and host cells transformed therewith |
| US5472692A (en) * | 1993-07-02 | 1995-12-05 | New England Deaconess Hospital Corporation | Pro-urokinase mutants |
| CA2157219C (en) * | 1994-08-31 | 2010-10-05 | Munehiro Noda | Process for purifying recombinant human serum albumin |
| US5760189A (en) * | 1995-06-02 | 1998-06-02 | Genetics Institute, Inc. | Protein recovery & purification methods |
| EP0833898A1 (en) | 1995-06-16 | 1998-04-08 | Novo Nordisk A/S | A process for producing trypsin (trypsinogen) |
| CN1187849A (zh) * | 1995-06-16 | 1998-07-15 | 诺沃挪第克公司 | 产生胰蛋白酶(胰蛋白酶原)的方法 |
| US5945328A (en) | 1995-06-16 | 1999-08-31 | Novo Nordisk A/S | Process for producing trypsin (trypsinogen) |
| ATE331798T1 (de) * | 1996-04-19 | 2006-07-15 | Jackson H M Found Military Med | Verfahren zur anregung einer immunantwort durch verabreichung von nutzorganismen, die intimin allein oder als fusionsprotein mit einem oder mehreren anderen antigenen exprimieren |
| GB9609702D0 (en) * | 1996-05-09 | 1996-07-10 | Royal Free Hosp School Med | Anticoagulant peptides |
| JP4515542B2 (ja) * | 1997-02-10 | 2010-08-04 | ジェネンテック， インコーポレイテッド | ヒレグリン変異体 |
| IL134653A0 (en) | 1997-08-22 | 2001-04-30 | Roche Diagnostics Gmbh | Zymogenic protease precursors that can be autocatalytically activated and their use |
| US20030207402A1 (en) | 1997-08-22 | 2003-11-06 | Erhard Kopetzki | Autocatalytically activatable zymogenic precursors of proteases and their use |
| US6159722A (en) * | 1997-12-03 | 2000-12-12 | Boehringer Mannheim Gmbh | Chimeric serine proteases |
| US6486303B1 (en) * | 1998-04-14 | 2002-11-26 | University Of Medicine & Dentistry Of New Jersey | Method for making hormone heterodimers |
| US6087558A (en) * | 1998-07-22 | 2000-07-11 | Prodigene, Inc. | Commercial production of proteases in plants |
| AU6388499A (en) * | 1998-09-21 | 2000-04-10 | Eli Lilly And Company | Production of soluble recombinant trypsinogen analogs |
| ES2265693T3 (es) * | 1998-10-16 | 2007-02-16 | Biogen Idec Ma Inc. | Proteinas de fusion con interferon-beta y usos. |
| EP1132479B1 (en) * | 1998-11-20 | 2009-04-22 | Fuso Pharmaceutical Industries Ltd. | Protein expression vector and utilization thereof |
| US20020102709A1 (en) * | 1999-02-19 | 2002-08-01 | Tetsuya Ishikawa | Collagen-binding physiologically active polypeptide |
| US6485957B1 (en) * | 1999-04-30 | 2002-11-26 | Ortho-Mcneil Pharmaceutical, Inc. | DNA encoding the human serine protease EOS |
| US6420157B1 (en) * | 1999-04-30 | 2002-07-16 | Ortho-Mcneil Pharmaceutical, Inc. | Zymogen activation system |
| US6458564B1 (en) * | 1999-08-31 | 2002-10-01 | Ortho-Mcneil Pharmaceutical, Inc. | DNA encoding the human serine protease T |
| AU7053500A (en) * | 1999-09-15 | 2001-04-17 | Eli Lilly And Company | Chymotrypsin-free trypsin |
| 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 |
| CA2408630A1 (en) * | 2000-06-19 | 2001-12-27 | Dyax Corp. | Novel enterokinase cleavage sequences |
| US6821755B2 (en) * | 2000-07-27 | 2004-11-23 | Boehringer Ingelheim International Gmbh | Preparation of a recombinant protein in a prokaryotic host cell |
| US6831059B2 (en) * | 2000-10-20 | 2004-12-14 | Allergan, Inc. | Compositions and methods for treating gonadotrophin related illnesses |
| US6632638B1 (en) * | 2000-11-17 | 2003-10-14 | Amgen, Inc. | Enhanced solubility of recombinant proteins using Uracil DNA glycosylase inhibitor |
| AU2002233230B2 (en) * | 2000-12-20 | 2007-02-01 | F. Hoffmann-La Roche Ag | Erythropoietin conjugates |
| WO2002061064A2 (de) * | 2001-02-01 | 2002-08-08 | Roche Diagnostics Gmbh | Verfahren zur herstellung von rekombinantem trypsin |
| CA2441378A1 (en) * | 2001-03-22 | 2002-10-03 | Dendreon San Diego Llc | Nucleic acid molecules encoding serine protease cvsp14, the encoded polypeptides and methods based thereon |
-
2002
- 2002-02-01 WO PCT/EP2002/001072 patent/WO2002061064A2/de not_active Ceased
- 2002-02-01 EP EP02716713A patent/EP1399568B1/de not_active Expired - Lifetime
- 2002-02-01 CA CA2437342A patent/CA2437342C/en not_active Expired - Fee Related
- 2002-02-01 AT AT02716713T patent/ATE310092T1/de active
- 2002-02-01 DE DE50204952T patent/DE50204952D1/de not_active Expired - Lifetime
- 2002-02-01 JP JP2002561621A patent/JP4159878B2/ja not_active Expired - Lifetime
- 2002-02-01 US US10/470,508 patent/US7276605B2/en not_active Expired - Lifetime
- 2002-02-01 CN CN02805619.1A patent/CN1545553B/zh not_active Expired - Lifetime
- 2002-02-01 CZ CZ20032337A patent/CZ303658B6/cs not_active IP Right Cessation
- 2002-02-01 DK DK02716713T patent/DK1399568T3/da active
-
2007
- 2007-09-11 US US11/853,483 patent/US7666629B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| WO2002061064A3 (de) | 2003-12-24 |
| CZ303658B6 (cs) | 2013-02-06 |
| CN1545553B (zh) | 2011-09-21 |
| US20040203095A1 (en) | 2004-10-14 |
| EP1399568A2 (de) | 2004-03-24 |
| JP4159878B2 (ja) | 2008-10-01 |
| DK1399568T3 (da) | 2006-03-27 |
| JP2004520049A (ja) | 2004-07-08 |
| CZ20032337A3 (cs) | 2004-04-14 |
| CA2437342A1 (en) | 2002-08-08 |
| DE50204952D1 (de) | 2005-12-22 |
| ATE310092T1 (de) | 2005-12-15 |
| US7666629B2 (en) | 2010-02-23 |
| EP1399568B1 (de) | 2005-11-16 |
| US20080064084A1 (en) | 2008-03-13 |
| CN1545553A (zh) | 2004-11-10 |
| US7276605B2 (en) | 2007-10-02 |
| WO2002061064A2 (de) | 2002-08-08 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKLA | Lapsed |
Effective date: 20190201 |