CA2210242A1 - Production of enzymatically active recombinant carboxypeptidase b - Google Patents
Production of enzymatically active recombinant carboxypeptidase bInfo
- Publication number
- CA2210242A1 CA2210242A1 CA002210242A CA2210242A CA2210242A1 CA 2210242 A1 CA2210242 A1 CA 2210242A1 CA 002210242 A CA002210242 A CA 002210242A CA 2210242 A CA2210242 A CA 2210242A CA 2210242 A1 CA2210242 A1 CA 2210242A1
- Authority
- CA
- Canada
- Prior art keywords
- procpb
- enzymatically active
- treating
- cpb
- purifying
- 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.)
- Granted
Links
- 102000003670 Carboxypeptidase B Human genes 0.000 title 1
- 108090000087 Carboxypeptidase B Proteins 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract 11
- 238000003776 cleavage reaction Methods 0.000 claims abstract 2
- 230000002255 enzymatic effect Effects 0.000 claims abstract 2
- 230000007017 scission Effects 0.000 claims abstract 2
- 210000004027 cell Anatomy 0.000 claims 3
- 238000004255 ion exchange chromatography Methods 0.000 claims 3
- 238000004587 chromatography analysis Methods 0.000 claims 2
- 230000002209 hydrophobic effect Effects 0.000 claims 2
- 230000003834 intracellular effect Effects 0.000 claims 2
- 239000006166 lysate Substances 0.000 claims 2
- 239000002244 precipitate Substances 0.000 claims 2
- 108010024636 Glutathione Proteins 0.000 claims 1
- 108010053070 Glutathione Disulfide Proteins 0.000 claims 1
- 241000392831 Pharyngostrongylus lambda Species 0.000 claims 1
- 102000004142 Trypsin Human genes 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 claims 1
- 210000002421 cell wall Anatomy 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 238000011026 diafiltration Methods 0.000 claims 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 claims 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 claims 1
- 239000013612 plasmid Substances 0.000 claims 1
- 230000003381 solubilizing effect Effects 0.000 claims 1
- 239000000126 substance Substances 0.000 claims 1
- 239000012588 trypsin Substances 0.000 claims 1
- 239000011592 zinc chloride Substances 0.000 claims 1
- 235000005074 zinc chloride Nutrition 0.000 claims 1
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims 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)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
Abstract
The subject invention provides a method of producing enzymatically active CPB
which comprises treating a recombinant cell containing DNA encoding ProCPB, so that the DNA directs expression of the ProCPB, recovering from the cell the ProCPB so expressed, treating the recovered ProCPB under conditions permitting folding of the ProCPB, subjecting the folded ProCPB to enzymatic cleavage to produce enzymatically active CPB and purifying the enzymatically active CPB.
which comprises treating a recombinant cell containing DNA encoding ProCPB, so that the DNA directs expression of the ProCPB, recovering from the cell the ProCPB so expressed, treating the recovered ProCPB under conditions permitting folding of the ProCPB, subjecting the folded ProCPB to enzymatic cleavage to produce enzymatically active CPB and purifying the enzymatically active CPB.
Claims (10)
1. A method of producing enzymatically active mammalian CPB which comprises:
(a) treating a recombinant cell containing DNA
encoding ProCPB, so that the DNA directs expression of the ProCPB;
(b) recovering from the cell the ProCPB so expressed;
(c) treating the recovered ProCPB under conditions permitting folding of the ProCPB;
(d) subjecting the folded ProCPB to enzymatic cleavage to produce enzymatically active CPB;
(e) purifying the enzymatically active CPB.
(a) treating a recombinant cell containing DNA
encoding ProCPB, so that the DNA directs expression of the ProCPB;
(b) recovering from the cell the ProCPB so expressed;
(c) treating the recovered ProCPB under conditions permitting folding of the ProCPB;
(d) subjecting the folded ProCPB to enzymatic cleavage to produce enzymatically active CPB;
(e) purifying the enzymatically active CPB.
2. A method according to claim 1 wherein the recovering of step (b) comprises:
(i) disrupting the cell wall of the recombinant cell to produce a lysate;
(ii) isolating intracellular precipitate from the lysate by centrifugation;
(iii) solubilizing the intracellular precipitate in a suitable buffer;
(i) disrupting the cell wall of the recombinant cell to produce a lysate;
(ii) isolating intracellular precipitate from the lysate by centrifugation;
(iii) solubilizing the intracellular precipitate in a suitable buffer;
3. A method according to claim 1 wherein the treating of step (c) comprises incubating the ProCPB at room temperature for a period of about 20-24 hours at a pH of about 9-9.5.
4. A method according to claim 1 wherein the treating of step (c) comprises incubating the ProCPB at room temperature for a period of about 20-24 hours at a pH of about 9-9.5 in the presence of ZnCl2, oxidized glutathione and reduced glutathione.
5. A method according to claim 1 wherein the subjecting of step (d) comprises:
(i) adjusting the pH to about 8.5; and (ii) cleaving the ProCPB with trypsin at 37°C for about 60 minutes.
(i) adjusting the pH to about 8.5; and (ii) cleaving the ProCPB with trypsin at 37°C for about 60 minutes.
6. A method according to claim 1 wherein the purifying of step (e) comprises ion-exchange chromatography.
7. A method according to claim 1 wherein the purifying of step (e) comprises ion-exchange chromatography and hydrophobic chromatography.
8. A method according to claim 1 wherein the purifying of step (e) comprises ion-exchange chromatography, hydrophobic chromatography and diafiltration.
9. A method according to claim 1 wherein the ProCPB
is expressed by plasmid p.lambda.ProCPB deposited under ATCC Accession No. 69673.
is expressed by plasmid p.lambda.ProCPB deposited under ATCC Accession No. 69673.
10. Enzymatically active mammalian CPB produced according to the method of claim 1, free of all other substances of mammalian origin.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US37823395A | 1995-01-25 | 1995-01-25 | |
| US08/378,233 | 1995-01-25 | ||
| PCT/US1996/000995 WO1996023064A1 (en) | 1995-01-25 | 1996-01-25 | Production of enzymatically active recombinant carboxypeptidase b |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2210242A1 true CA2210242A1 (en) | 1996-08-01 |
| CA2210242C CA2210242C (en) | 2010-04-27 |
Family
ID=23492292
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2210242A Expired - Lifetime CA2210242C (en) | 1995-01-25 | 1996-01-25 | Production of enzymatically active recombinant carboxypeptidase b |
Country Status (22)
| Country | Link |
|---|---|
| US (1) | US5948668A (en) |
| EP (1) | EP0871718B1 (en) |
| JP (1) | JP4250716B2 (en) |
| KR (1) | KR100566136B1 (en) |
| CN (1) | CN1144874C (en) |
| AT (1) | ATE358717T1 (en) |
| AU (1) | AU698889B2 (en) |
| BR (1) | BR9606795A (en) |
| CA (1) | CA2210242C (en) |
| CZ (1) | CZ292541B6 (en) |
| DE (1) | DE69637011T2 (en) |
| DK (1) | DK0871718T3 (en) |
| ES (1) | ES2284167T3 (en) |
| HK (1) | HK1014986A1 (en) |
| HU (1) | HU225673B1 (en) |
| IL (1) | IL116696A (en) |
| MX (1) | MX9705279A (en) |
| NZ (1) | NZ302874A (en) |
| PL (1) | PL183228B1 (en) |
| PT (1) | PT871718E (en) |
| WO (1) | WO1996023064A1 (en) |
| ZA (1) | ZA96515B (en) |
Families Citing this family (24)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH11511970A (en) | 1995-08-16 | 1999-10-19 | ゼネカ・リミテッド | Chemical compound |
| DK2316475T3 (en) | 1998-08-06 | 2017-11-20 | Mountain View Pharmaceuticals Inc | Isolated tetrameric uricase |
| US20040117863A1 (en) * | 1998-09-18 | 2004-06-17 | Edge Michael D. | Transgenically produced fusion proteins |
| DE19915938A1 (en) | 1999-04-09 | 2000-10-19 | Aventis Pharma Gmbh | Production of pancreatic procarboxypeptidase B, isoforms and muteins thereof and their use |
| EP1220864A4 (en) * | 1999-09-17 | 2003-05-21 | Genzyme Transgenics Corp | Transgenically produced fusion proteins |
| EP1632575B1 (en) * | 1999-09-29 | 2009-01-28 | Lexicon Pharmaceuticals, Inc. | Human carboxypeptidases and polynucleotides encoding the same |
| DE60023928T2 (en) | 1999-09-29 | 2006-07-27 | Lexicon Genetics Inc., The Woodlands | HUMAN CARBOXYPEPTIDASES AND THESE CODING POLYNUCLEOTIDES |
| AU2001229253A1 (en) * | 2000-01-12 | 2001-07-24 | Eli Lilly And Company | Carboxypeptidase b free of animal products and contaminating enyzme activity |
| EP1538203B1 (en) * | 2003-12-05 | 2010-01-20 | Roche Diagnostics GmbH | Recombinantly expressed carboxypeptidase B and purification thereof |
| ES2337684T3 (en) * | 2003-12-05 | 2010-04-28 | F. Hoffmann-La Roche Ag | RECOMBINANT CARBOXIPEPTIDASE B AND ITS PURIFICATION. |
| DK1794294T3 (en) * | 2004-09-27 | 2011-10-31 | Sanofi Aventis Deutschland | Recombinant carboxypeptidase B |
| LT3321359T (en) | 2005-04-11 | 2021-05-10 | Horizon Pharma Rheumatology Llc | Variant forms of urate oxidase and use thereof |
| US20080159976A1 (en) * | 2005-04-11 | 2008-07-03 | Jacob Hartman | Methods for lowering elevated uric acid levels using intravenous injections of PEG-uricase |
| US8148123B2 (en) | 2005-04-11 | 2012-04-03 | Savient Pharmaceuticals, Inc. | Methods for lowering elevated uric acid levels using intravenous injections of PEG-uricase |
| CN101194016B (en) | 2005-04-11 | 2012-09-05 | 萨文特医药公司 | A variant form of urate oxidase and use thereof |
| WO2006125452A1 (en) * | 2005-05-23 | 2006-11-30 | Universite De Geneve | Injectable superparamagnetic nanoparticles for treatment by hyperthermia and use for forming an hyperthermic implant |
| PL2013225T3 (en) | 2006-04-12 | 2015-06-30 | Crealta Pharmaceuticals Llc | Purification of proteins with cationic surfactant |
| CN101058805B (en) * | 2007-03-21 | 2011-09-07 | 北京贯虹科技有限公司 | Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B |
| US8993714B2 (en) * | 2007-10-26 | 2015-03-31 | Imiplex Llc | Streptavidin macromolecular adaptor and complexes thereof |
| US9102526B2 (en) | 2008-08-12 | 2015-08-11 | Imiplex Llc | Node polypeptides for nanostructure assembly |
| WO2010132363A1 (en) | 2009-05-11 | 2010-11-18 | Imiplex Llc | Method of protein nanostructure fabrication |
| WO2010151823A1 (en) | 2009-06-25 | 2010-12-29 | Savient Pharmaceuticals Inc. | Methods and kits for predicting infusion reaction risk and antibody-mediated loss of response by monitoring serum uric acid during pegylated uricase therapy |
| CN101967467B (en) * | 2009-07-28 | 2012-11-14 | 上海雅心生物技术有限公司 | Production and application of high-stability recombination carboxypeptidase B |
| US11918624B2 (en) * | 2020-06-10 | 2024-03-05 | Kelsius Laboratories LLC | Therapeutic composition for use in the treatment of COVID-19 and other cytokine storm associated disorders |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE724843A (en) * | 1967-12-04 | 1969-06-03 | ||
| US4511503A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| HU190129B (en) * | 1983-07-11 | 1986-08-28 | Reanal Finomvegyszergyar,Hu | Process for the isolation of carboxypeptidase b enzyme from mammal pancreas |
| US5206161A (en) * | 1991-02-01 | 1993-04-27 | Genentech, Inc. | Human plasma carboxypeptidase B |
| FR2692907B1 (en) * | 1992-06-25 | 1995-06-30 | Rhone Poulenc Rorer Sa | MODIFIED KLUYVEROMYCES YEASTS, PREPARATION AND USE. |
| JP3472587B2 (en) * | 1992-08-28 | 2003-12-02 | アベンティス ファーマ株式会社 | Bone-related carboxypeptidase-like protein and method for producing the same |
| JPH09505998A (en) * | 1993-11-16 | 1997-06-17 | イーライ・リリー・アンド・カンパニー | DNA sequence encoding porcine pancreatic carboxypeptidase B |
-
1996
- 1996-01-08 IL IL11669696A patent/IL116696A/en not_active IP Right Cessation
- 1996-01-23 ZA ZA96515A patent/ZA96515B/en unknown
- 1996-01-25 EP EP96905218A patent/EP0871718B1/en not_active Expired - Lifetime
- 1996-01-25 JP JP52300096A patent/JP4250716B2/en not_active Expired - Lifetime
- 1996-01-25 BR BR9606795A patent/BR9606795A/en not_active Application Discontinuation
- 1996-01-25 HU HU9800091A patent/HU225673B1/en unknown
- 1996-01-25 CA CA2210242A patent/CA2210242C/en not_active Expired - Lifetime
- 1996-01-25 WO PCT/US1996/000995 patent/WO1996023064A1/en active IP Right Grant
- 1996-01-25 ES ES96905218T patent/ES2284167T3/en not_active Expired - Lifetime
- 1996-01-25 AU AU49034/96A patent/AU698889B2/en not_active Expired
- 1996-01-25 PL PL96321499A patent/PL183228B1/en unknown
- 1996-01-25 CN CNB961923679A patent/CN1144874C/en not_active Expired - Lifetime
- 1996-01-25 DE DE69637011T patent/DE69637011T2/en not_active Expired - Lifetime
- 1996-01-25 DK DK96905218T patent/DK0871718T3/en active
- 1996-01-25 CZ CZ19972258A patent/CZ292541B6/en not_active IP Right Cessation
- 1996-01-25 AT AT96905218T patent/ATE358717T1/en active
- 1996-01-25 KR KR1019970705046A patent/KR100566136B1/en not_active IP Right Cessation
- 1996-01-25 NZ NZ302874A patent/NZ302874A/en not_active IP Right Cessation
- 1996-01-25 PT PT96905218T patent/PT871718E/en unknown
-
1997
- 1997-01-13 US US08/782,760 patent/US5948668A/en not_active Expired - Lifetime
- 1997-07-11 MX MX9705279A patent/MX9705279A/en unknown
-
1998
- 1998-12-30 HK HK98119240A patent/HK1014986A1/en not_active IP Right Cessation
Also Published As
| Publication number | Publication date |
|---|---|
| ATE358717T1 (en) | 2007-04-15 |
| BR9606795A (en) | 1997-12-30 |
| CN1177377A (en) | 1998-03-25 |
| CZ292541B6 (en) | 2003-10-15 |
| EP0871718B1 (en) | 2007-04-04 |
| KR100566136B1 (en) | 2006-11-10 |
| AU4903496A (en) | 1996-08-14 |
| KR19980701652A (en) | 1998-06-25 |
| HUP9800091A3 (en) | 2004-03-29 |
| US5948668A (en) | 1999-09-07 |
| NZ302874A (en) | 1998-11-25 |
| CA2210242C (en) | 2010-04-27 |
| DE69637011D1 (en) | 2007-05-16 |
| JP4250716B2 (en) | 2009-04-08 |
| EP0871718A1 (en) | 1998-10-21 |
| JPH11503002A (en) | 1999-03-23 |
| IL116696A0 (en) | 1996-05-14 |
| CN1144874C (en) | 2004-04-07 |
| HK1014986A1 (en) | 1999-10-08 |
| ZA96515B (en) | 1996-08-15 |
| AU698889B2 (en) | 1998-11-12 |
| PL321499A1 (en) | 1997-12-08 |
| WO1996023064A1 (en) | 1996-08-01 |
| DK0871718T3 (en) | 2007-07-02 |
| PT871718E (en) | 2007-06-26 |
| HUP9800091A2 (en) | 1998-05-28 |
| ES2284167T3 (en) | 2007-11-01 |
| CZ225897A3 (en) | 1998-10-14 |
| HU225673B1 (en) | 2007-06-28 |
| DE69637011T2 (en) | 2007-12-13 |
| MX9705279A (en) | 1998-06-30 |
| EP0871718A4 (en) | 2000-06-07 |
| PL183228B1 (en) | 2002-06-28 |
| IL116696A (en) | 1999-08-17 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| MKEX | Expiry |
Effective date: 20160125 |