WO1995020039B1 - Customized proteases with altered transacylation activity - Google Patents
Customized proteases with altered transacylation activityInfo
- Publication number
- WO1995020039B1 WO1995020039B1 PCT/US1995/006682 US9506682W WO9520039B1 WO 1995020039 B1 WO1995020039 B1 WO 1995020039B1 US 9506682 W US9506682 W US 9506682W WO 9520039 B1 WO9520039 B1 WO 9520039B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- carboxypeptidase
- customized
- amino acid
- preselected
- modified
- 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.)
- Ceased
Links
Abstract
The invention provides customized proteases (i.e., mutant enzymes), e.g. carboxypeptidase Y, methods of making customized proteases, as well as methods of using customized proteases. The customized proteases of the invention are derived from the known proteases. Altered transacylation reactions include the capability to perform transacylation reactions not substantially catalyzed by the known protease or the capability to perform transacylation reactions with improved yields, or both. The methods of the invention provide for customized proteases through site specific or random mutagenesis of the active site amino acids of the known proteases. The invention also provides for methods of using the customized proteases to prepare a preselected transacylation products. The preselected transacylation products produced can be modified by substitution at the N- or C-terminal with nucleophiles such as L-amino acids, D-amino acids, amino acid amides, and radioactive amino acids.
Claims
1. A customized carboxypeptidase derived from a known carboxypeptidase, wherein the customized carboxypeptidase has an active site modified by substitution of at least one amino acid residue that corresponds in the active site to one of the following residues in the active site of carboxypeptidase Y: Pro54, Tyr1 7, Tyrl85, Tyr188, Glu215, Arg216, Asn241, eu245, Trp312, Ile340. Cys341, Tyr49, Asn51, Gly52, Cys56, Thr60, Phe64, Glu65, Glul45, Tyr256, Tyr269, Leu272, Ser297 and Cys 298, and is capable of catalyzing a transacylation reaction in which a preselected substrate is modified with a preselected nucleophile, and wherein the same transacylation reaction is not substantially catalyzed by the known protease.
2. A customized carboxypeptidase derived from a known carboxypeptidase, wherein the customized carboxypeptidase has an active site modified by substitution of at least one amino acid in each of the Sx and S1 ' binding sites.
3. The customized carboxypeptidase of claim 2, wherein said at least one amino acid is modified by substitution in the S-^ binding site of the carboxypeptidase corresponds to at least one of the following amino acid residues of the Sx binding site of carboxypeptidase Y: Pro54, Tyrl47, Tyrl85, Tyrl88, Glu215, Arg216, Asn241, Leu245, Trp312, Ile340 and Cys341, Leul78; and wherein the at least one amino acid modified by substitution in the S binding site of the carboxypeptidase corresponds to at least one of the following amino acid residues of the S1 binding site of carboxypeptidase Y: Tyr49, Asn51, Gly52, Cys56, Thr60, Phe64, Glu65, Glul45, Tyr256, Tyr269, Leu272, Ser297, Cys298 and Met398.
4. The customized carboxypeptidase of claim 3, having 1 06
active site amino acid residues corresponding to Leul78 and Met398 of carboxypeptidase Y modified by substitution.
5. The customized carboxypeptidase of claim 4, wherein said amino acid residue corresponding to eul78 is replaced with Ser and said amino acid residue corresponding to Metl78 is replaced with Leu.
6. The customized carboxypeptidase of claim 1, wherein said preselected nucleophile is selected from the group consisting of an acidic amino acid amide, a basic amino acid amide, a peptide amide, an amino acid and an amino acid ester.
7. The customized carboxypeptidase of claim 1, wherein said preselected substrate has a basic or acidic penultimate amino acid.
8. The customized carboxypeptidase of claim 1, wherein said preselected substrate corresponds to GRF (1-43) -Ala and said preselected nucleophile is a leucine amide.
9. A method for preparing a customized carboxypeptidase derived from a known carboxypeptidase sequence and that has a modified active site and that functions to alter the transacylating capability of the known protease comprising the steps of:
(a) providing a DNA sequence that encodes the known protease; (b) modifying at least one codon for at least one amino acid residue in each of the Sx and S1 ' portions of the active site to form a mutant DNA sequence, wherein the mutant DNA sequence encodes a customized protease with an altered transacylation capability; and (c) transforming a suitable host cell with the mutant DNA sequence to provide for expression of the customized protease. 1 07
10. A process for modifying a preselected substrate by transacylation using a customized carboxypeptidase derived from a known carboxypeptidase sequence comprising: incubating a customized carboxypeptidase with a preselected substrate and a preselected nucleophile to form a preselected transacylation product, wherein the customized carboxypeptidase has a modified active site and wherein the preselected substrate and the preselected nucleophile are not substantially transacylated by the known carboxypeptidase.
11. The method of claim 10, wherein the preselected nucleophile is a leucine amide, the preselected substrate is growth hormone releasing factor with a c-terminal alanine, and the preselected transacylation product is GRF(l-44) -NH2.
12. The method of claim 10, wherein the customized carboxypeptidase has an active site modified by substitution of at least one amino acid residue corresponding to the following active site residues of carboxypeptidase Y: Pro54, Leul78 and et398 ;and wherein the customized carboxypeptidase is capable of catalyzing a transacylation reaction in which a preselected substrate is modified with a preselected nucleophile, and wherein the same transacylation reaction is not substantially catalyzed by the known carboxypeptidas .
13. The method of claim 10, wherein the customized carboxypeptidase is modified to have a Ser at the amino acid residue corresponding to Leul78 of carboxypeptidase Y and a Leu at the amino acid residue corresponding to Met398 of carboxypeptidase Y.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU26513/95A AU679861B2 (en) | 1993-10-28 | 1994-10-28 | Customized proteases with altered transacylation activity |
| JP7517672A JPH09504438A (en) | 1993-10-28 | 1994-10-28 | Special protease with modified acyl transfer activity |
| NZ283995A NZ283995A (en) | 1993-10-28 | 1994-10-28 | Customized (mutant) carboxypeptidase Y having modified S1 subsite |
| EP95921432A EP0723585A1 (en) | 1993-10-28 | 1994-10-28 | Customized proteases |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US14470493A | 1993-10-28 | 1993-10-28 | |
| US08/144,704 | 1993-10-28 | ||
| US08/329,892 US6187579B1 (en) | 1993-10-28 | 1994-10-27 | Customized proteases |
| US08/329,892 | 1994-10-27 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO1995020039A2 WO1995020039A2 (en) | 1995-07-27 |
| WO1995020039A3 WO1995020039A3 (en) | 1996-03-21 |
| WO1995020039B1 true WO1995020039B1 (en) | 1996-05-30 |
Family
ID=26842276
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/US1995/006682 Ceased WO1995020039A2 (en) | 1993-10-28 | 1994-10-28 | Customized proteases with altered transacylation activity |
Country Status (7)
| Country | Link |
|---|---|
| US (2) | US6187579B1 (en) |
| EP (1) | EP0723585A1 (en) |
| JP (1) | JPH09504438A (en) |
| AU (1) | AU679861B2 (en) |
| CA (1) | CA2174525A1 (en) |
| NZ (1) | NZ283995A (en) |
| WO (1) | WO1995020039A2 (en) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
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| GB9323429D0 (en) * | 1993-11-12 | 1994-01-05 | Wellcome Found | Therapy |
| PL325017A1 (en) * | 1995-08-16 | 1998-07-06 | Zeneca Ltd | Chemical compounds |
| DE19535082A1 (en) | 1995-09-21 | 1997-03-27 | Henkel Ecolab Gmbh & Co Ohg | Paste-like detergent and cleaning agent |
| US20020164580A1 (en) * | 1995-12-07 | 2002-11-07 | Diversa Corporation | Combinatorial screening of mixed populations of organisms |
| US6537776B1 (en) * | 1999-06-14 | 2003-03-25 | Diversa Corporation | Synthetic ligation reassembly in directed evolution |
| US6764835B2 (en) * | 1995-12-07 | 2004-07-20 | Diversa Corporation | Saturation mutageneis in directed evolution |
| US6358709B1 (en) | 1995-12-07 | 2002-03-19 | Diversa Corporation | End selection in directed evolution |
| US6238884B1 (en) | 1995-12-07 | 2001-05-29 | Diversa Corporation | End selection in directed evolution |
| US6740506B2 (en) | 1995-12-07 | 2004-05-25 | Diversa Corporation | End selection in directed evolution |
| US7018793B1 (en) * | 1995-12-07 | 2006-03-28 | Diversa Corporation | Combinatorial screening of mixed populations of organisms |
| US6352842B1 (en) | 1995-12-07 | 2002-03-05 | Diversa Corporation | Exonucease-mediated gene assembly in directed evolution |
| US6171820B1 (en) | 1995-12-07 | 2001-01-09 | Diversa Corporation | Saturation mutagenesis in directed evolution |
| US20040023327A1 (en) * | 1995-12-07 | 2004-02-05 | Short Jay M. | End selection in directed evolution |
| US6361974B1 (en) | 1995-12-07 | 2002-03-26 | Diversa Corporation | Exonuclease-mediated nucleic acid reassembly in directed evolution |
| US6939689B2 (en) | 1995-12-07 | 2005-09-06 | Diversa Corporation | Exonuclease-mediated nucleic acid reassembly in directed evolution |
| US20030219752A1 (en) * | 1995-12-07 | 2003-11-27 | Diversa Corporation | Novel antigen binding molecules for therapeutic, diagnostic, prophylactic, enzymatic, industrial, and agricultural applications, and methods for generating and screening thereof |
| US6713279B1 (en) | 1995-12-07 | 2004-03-30 | Diversa Corporation | Non-stochastic generation of genetic vaccines and enzymes |
| US5830696A (en) | 1996-12-05 | 1998-11-03 | Diversa Corporation | Directed evolution of thermophilic enzymes |
| US6562594B1 (en) | 1999-09-29 | 2003-05-13 | Diversa Corporation | Saturation mutagenesis in directed evolution |
| DE19636035A1 (en) | 1996-09-05 | 1998-03-12 | Henkel Ecolab Gmbh & Co Ohg | Paste-like detergent and cleaning agent |
| WO1998020156A1 (en) * | 1996-11-04 | 1998-05-14 | Merck Frosst Canada & Co. | Hydrolase binding assay |
| DE19703364A1 (en) | 1997-01-30 | 1998-08-06 | Henkel Ecolab Gmbh & Co Ohg | Paste-like detergent and cleaning agent |
| US5985627A (en) * | 1997-02-28 | 1999-11-16 | Carlsberg Laboratory | Modified carboxypeptidase |
| US6908757B1 (en) | 1998-03-26 | 2005-06-21 | The Procter & Gamble Company | Serine protease variants having amino acid deletions and substitutions |
| US6461834B1 (en) * | 1998-11-06 | 2002-10-08 | Bionebraska, Inc. | Clostripain catalyzed amidation of peptides |
| DE19857687A1 (en) | 1998-12-15 | 2000-06-21 | Henkel Ecolab Gmbh & Co Ohg | Pasty detergent |
| US20090130718A1 (en) * | 1999-02-04 | 2009-05-21 | Diversa Corporation | Gene site saturation mutagenesis |
| KR20020021398A (en) | 1999-07-22 | 2002-03-20 | 데이비드 엠 모이어 | Subtilisin protease variants having amino acid substitutions in defined epitope regions |
| CZ2002171A3 (en) | 1999-07-22 | 2002-06-12 | The Procter & Gamble Company | Proteinase conjugate, cleansing agent and preparation for personal hygiene |
| US6946128B1 (en) | 1999-07-22 | 2005-09-20 | The Procter & Gamble Company | Protease conjugates having sterically protected epitope regions |
| CA2379712A1 (en) | 1999-07-22 | 2001-02-01 | The Procter & Gamble Company | Subtilisin protease variants having amino acid deletions and substitutions in defined epitope regions |
| CN1100789C (en) * | 2000-01-13 | 2003-02-05 | 中国人民解放军第二军医大学 | Preparation process of gene recomibination calcitonin or calcitonin analog |
| US20030170846A1 (en) * | 2000-04-24 | 2003-09-11 | Weisgerber David John | Enzyme variants having one or more D-amino acid substitutions |
| ES2261491T3 (en) * | 2000-09-04 | 2006-11-16 | Cellpep S.A. | DERIVATIVES OF THE MAUROTOXINA, PI1 AND HSTX1. |
| US20040009498A1 (en) * | 2002-01-14 | 2004-01-15 | Diversa Corporation | Chimeric antigen binding molecules and methods for making and using them |
| US20030219854A1 (en) * | 2002-03-21 | 2003-11-27 | Micrologix Biotech Inc. | Methods for producing modified anti-infective peptides |
| US20040171070A1 (en) * | 2002-05-20 | 2004-09-02 | Ramagauri Bhikhabhai | Peptide analysis using a solid support |
| CA2569707C (en) * | 2004-08-13 | 2013-09-24 | F. Hoffmann-La Roche Ag | C-terminal modification of polypeptides |
| JP5114201B2 (en) * | 2004-09-27 | 2013-01-09 | サノフイ−アベンテイス・ドイチユラント・ゲー・エム・ベー・ハー | Recombinant carboxypeptidase B |
| WO2007082890A1 (en) * | 2006-01-17 | 2007-07-26 | N.V. Organon | SELECTIVE ENZYMATIC HYDROLYSIS OF C-TERMINAL tert-BUTYL ESTERS OF PEPTIDES |
| US9133500B2 (en) * | 2009-08-10 | 2015-09-15 | MorphoSys A6 | Screening strategies for the identification of binders |
| BR112014027861A2 (en) | 2012-05-11 | 2017-12-12 | Novozymes As | method of preparation of a must |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6339237B2 (en) | 1979-04-06 | 1988-08-04 | Karuruberugu Baiotekunorojii Ltd As | |
| DK149824C (en) | 1982-01-22 | 1987-03-16 | Carlsberg Biotechnology Ltd | PROCEDURE FOR ENZYMATIC REPLACEMENT OF B-30 AMINO ACIDS IN INSULINES |
| US5185258A (en) | 1984-05-29 | 1993-02-09 | Genencor International, Inc. | Subtilisin mutants |
| US4959312A (en) * | 1985-05-31 | 1990-09-25 | The University Of Tennessee Research Corporation | Full spectrum mutagenesis |
| JPH07121222B2 (en) | 1987-03-24 | 1995-12-25 | 寶酒造株式会社 | Chemically modified serine protease-containing composition, its production method and use |
| GB8807271D0 (en) | 1988-03-26 | 1988-04-27 | Preece A W | Cell fusion apparatus |
| US5049656A (en) | 1988-12-21 | 1991-09-17 | Board Of Regents Of The University Of Nebraska | Sequential peptide and oligonucleotide syntheses using immunoaffinity techniques |
| EP0542931B1 (en) | 1990-08-09 | 1999-12-08 | Genentech, Inc. | Serine protease variants having peptide ligase activity |
-
1994
- 1994-10-27 US US08/329,892 patent/US6187579B1/en not_active Expired - Fee Related
- 1994-10-28 AU AU26513/95A patent/AU679861B2/en not_active Ceased
- 1994-10-28 NZ NZ283995A patent/NZ283995A/en unknown
- 1994-10-28 WO PCT/US1995/006682 patent/WO1995020039A2/en not_active Ceased
- 1994-10-28 CA CA002174525A patent/CA2174525A1/en not_active Abandoned
- 1994-10-28 JP JP7517672A patent/JPH09504438A/en not_active Ceased
- 1994-10-28 EP EP95921432A patent/EP0723585A1/en not_active Withdrawn
-
1997
- 1997-07-23 US US08/899,324 patent/US5945329A/en not_active Expired - Fee Related
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