Claims
- 1. A method for making a ligation product comprising the step of contacting a subtilisin-type serine protease variant having peptide ligase activity with a first and a second ligation substrate, wherein said first ligation substrate comprises at least an R1 residue where the carboxy-terminus of said R1 residue is esterified with an organic alcohol or thiol, said second ligation substrate comprises at least an R1' residue and said serine protease variant has an amino acid sequence not found in nature which is derived from a precursor subtilisin-type serine protease having an .alpha.-helix containing proline at a residue equivalent to proline 225 in Bacillus amyloliquefaciens subtilisin and a catalytic serine at or near the amino terminus of said .alpha.-helix equivalent to serine 221 in Bacillus amyloliquefaciens subtilisin, said derivation comprising:
- a) replacing said catalytic serine with a first amino acid having a different nucleophilic side chain or modifying the side chain of said catalytic serine, to substitute the nucleophilic oxygen of said side chain with a nucleophilic sulfur, thereby converting said catalytic serine to cysteine, and
- b) replacing said proline with a second different amino acid comprising a helix-forming amino acid, or replacing said proline with a second different amino acid having a side chain volume less than the side chain volume of proline;
- said serine protease variant being characterized by having peptide ligase activity in aqueous solution which is greater than that of said precursor serine protease variant containing only said substitution or modification of said nucleophilic oxygen.
- 2. The method of claim 1 wherein the organic moiety of said alcohol or thiol is selected from the group consisting of
- C.sub.6 -C.sub.12 aryl where the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), C.sub.1 -C.sub.8 alkyl, halo-C.sub.1 -C.sub.8 alkyl, C.sub.1 -C.sub.8 -alkoxy, amino, phenyloxy, phenyl, acetamido, benzamido, di-C.sub.1 -C.sub.8 alkylamino, C.sub.1 -C.sub.8 alkylamino, C.sub.6 -C.sub.12 aroyl, C.sub.1 -C.sub.8 alkanoyl, and hydroxy-C.sub.1 -C.sub.8 alkyl,
- C.sub.1 -C.sub.12 alkyl either substituted or unsubstituted, branched, straight chain or cyclo where the substituents are selected from halo (F, Cl, Br, I),
- C.sub.1 -C.sub.8 alkoxy,
- C.sub.6 -C.sub.12 aryloxy where the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), C.sub.1 -C.sub.8 alkyl, C.sub.1 -C.sub.8 -alkoxy, amino, phenyloxy, acetamido, benzamido, di-C.sub.1 -C.sub.8 alkylamino, C.sub.1 -C.sub.8 alkylamino, C.sub.6 -C.sub.12 aroyl, and C.sub.1 -C.sub.8 alkanoyl,
- isothioureido,
- C.sub.3 -C.sub.7 cycloalkyl,
- ureido,
- amino,
- C.sub.1 -C.sub.8 alkylamino,
- di-C.sub.1 -C.sub.8 alkylamino,
- hydroxy,
- amino-C.sub.2 -C.sub.8 alkylthio,
- amino-C.sub.2 -C.sub.8 alkoxy,
- acetamido,
- benzamido wherein the phenyl ring is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo (F, Cl, Br, I), C.sub.1 -C.sub.8 alkyl, C.sub.1 -C.sub.8 -alkoxy, amino, phenyloxy, acetamido, benzamido, di-C.sub.1 -C.sub.8 alkylamino, C.sub.1 -C.sub.8 alkylamino, C.sub.6 -C.sub.12 aroyl, C.sub.1 -C.sub.8 alkanoyl,
- C.sub.6 -C.sub.12 arylamino wherein the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo, C.sub.1 -C.sub.8 alkyl, C.sub.1 -C.sub.8 -alkoxy, amino, phenyloxy, acetamido, benzamido, di-C.sub.1 -C.sub.8 alkylamino, C.sub.1 -C.sub.8 alkylamino, C.sub.6 -C.sub.12 aroyl, and C.sub.1 -C.sub.8 alkanoyl,
- guanidino,
- phthalimido,
- mercapto,
- C.sub.1 -C.sub.8 alkylthio,
- C.sub.6 -C.sub.12 arylthio,
- carboxy
- carboxamide,
- carbo-C.sub.1 -C.sub.8 alkoxy,
- C.sub.6 -C aryl wherein the aryl group is unsubstituted or substituted by one or more of the groups nitro, hydroxy, halo, C.sub.1 -C.sub.8 alkyl, C.sub.1 -C.sub.8 -alkoxy, amino, phenyloxy, acetamido, benzamido, di-C.sub.1 -C.sub.8 alkylamino, C.sub.1 -C.sub.8 alkylamino, hydroxy-C.sub.1 -C.sub.8 alkyl, C.sub.6 -C.sub.12 aroyl, and C.sub.1 -C.sub.8 alkanoyl, and
- aromatic heterocycle wherein the heterocyclic groups have 5-10 ring atoms and contain up to two O, N, or S heteroatoms, and 2-hydroxy carboxylic acid.
- 3. The method of claim 1 wherein the carboxy terminus of said R1 residue is esterified with a 2-hydroxy carboxylic acid.
- 4. The method of claim 3 wherein said 2-hydroxy carboxylic acid is glycolate or lactate.
- 5. The method of claim 3 wherein said 2-hydroxy carboxylic acid is glycolate.
- 6. The method of claim 3 wherein said 2-hydroxy carboxylic acid is amidated.
- 7. The method of claim 6 wherein said 2-hydroxy carboxylic acid is amidated with the amino-terminus of an amino acid or third peptide.
- 8. The method of claim 6 wherein the 2-hydroxy carboxylic acid is selected from the group glc-amide, glc-Phe-amide, glc-Phe-Gly-amide, lac-amide, lac-Leu-amide, lac-Phe-amide and Sbz.
- 9. The method of claim 1 wherein the R and R' residues of each of said first and said second ligation substrates respectively has a length of between 1 and 25 residues and said ligation product contains about 50 residues.
- 10. The method of claim 1 wherein said R1 amino acid residue of said first ligation peptide is a large hydrophobic amino acid selected from the group consisting of phenylalanine, tyrosine, tryptophan, leucine, and methionine.
- 11. The method of claim 1 wherein said R1' residue is selected from the group consisting of amino acid residues not consisting of isoleucene and proline.
- 12. The method of claim 1 wherein said second ligation substrate comprises at least R1' and R2' residues and said R2' residue is selected from the group consisting of phenylalanine, tyrosine, tryptophan, leucine, methionine, arginine and lysine.
- 13. The method of claim 1 wherein said contacting is under conditions that allow the C-terminus of said first ligation substrate and the N-terminus of said second ligation substrate to be in the correct conformation for ligation to occur, without substantially destroying the ligation activity of said serine protease variant.
- 14. The method of claim 1 wherein said first ligation substrate comprises at least one component that is not a naturally occurring amino acid such that said component is incorporated into said ligation product.
- 15. The method of claim 14 wherein said component is an unnatural amino acid analog.
- 16. The method of claim 14 wherein said component is isotopically labeled.
- 17. The method of claim 14 wherein said component not proteinaceous.
- 18. The method of claim 1 wherein said second ligation substrate comprises at least one component that is not a naturally occurring amino acid such that said component is incorporated into said ligation product.
- 19. The method of claim 18 wherein said component is an unnatural amino acid analog.
- 20. The method of claim 18 wherein said component is isotopically labeled.
- 21. The method of claim 18 wherein said component not proteinaceous.
- 22. A method for making a polypeptide comprising:
- ligating a first and second ligation substrate with a subtilisin-type serine protease variant having peptide ligase activity, wherein said serine protease variant has an amino acid sequence not found in nature which is derived from a precursor subtilisin-type serine protease by changing at least two amino acid residues in said precursor having an .alpha.-helix containing proline at a residue equivalent to proline 225 in Bacillus amyloliquefaciens subtilisin and a catalytic serine at or near the amino terminus of said .alpha.-helix equivalent to serine 221 in Bacillus amyloliquefaciens subtilisin, said derivation comprising:
- a) replacing said catalytic serine with a first amino acid having a different nucleophilic side chain or modifying the side chain of said catalytic serine, to substitute the nucleophilic oxygen of said side chain with a nucleophilic sulfur, thereby converting said catalytic serine to cysteine, and
- b) replacing said proline with a second different amino acid comprising a helix-forming amino acid, or replacing said proline with a second different amino acid having a side chain volume less than the side chain volume of proline;
- said serine protease variant being characterized by having peptide ligase activity in aqueous solution which is greater than that of said precursor serine protease variant containing only said substitution or modification of said nucleophilic oxygen, wherein said first ligation substrate comprises at least an amino terminus and a carboxy terminus amino acid residue, the carboxy terminus of the carboxy terminus residue being bonded to a carboxy terminus activating group (CTAG), the amino terminus of the amino terminus residue being bonded to an amino terminus protecting group (ATPG), and wherein the second ligation substrate comprises at least two amino acid residues having a free amino terminus, under conditions suitable for formation of a ligation product; and
- deprotecting the amino terminus of the ligation product thereby forming a deprotected ligation product.
- 23. The method of claim 22 further comprising sequentially repeating the ligating and deprotecting steps one or more times with the proviso that the deprotected ligation product of the previous step becomes the second ligation substrate in the subsequent ligating step.
- 24. The method of claim 22 wherein the first ligation substrate contains from about 10 to about 30 amino acid residues.
- 25. The method of claim 22 wherein the carboxy terminus activating group is a glycolate(glc) or lactate(lac) optionally derivatized with an amino group, or from one to three naturally occurring .alpha.-amino acid residues, the carboxy terminus of the terminal amino acid residue being optionally derivatized with an amino group.
- 26. The method of claim 22 wherein the carboxy terminus activating group is selected from the group: glc-amide, glc-Phe-amide, glc-Phe-Gly-amide, lac-amide, lac-Leu-amide, lac-Phe-amide and Sbz.
- 27. The method of claim 13 wherein the amino terminus protecting group is selected from the group consisting of: t-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyloxycarbonyl, p-methoxybenzyloxycarbonyl, benzyloxycarbonyl, halobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl, isopropyloxycarbonyl, isonicotinyl(iNOC), phenylacetic acid, and pyridine acetic acid.
Parent Case Info
This application is a divisional of Ser. No. 08/218,873, filed 28 Mar. 1994, which is a continuation of Ser. No. 08/013,445, filed 4 Feb. 1993, which is a continuation-in-part of PCT/US91/05480, filed 6 Aug. 1991, which is a continuation-in-part of Ser. No. 07/566,026, filed 9 Aug. 1990, each abandoned.
US Referenced Citations (2)
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4760025 |
Estell et al. |
Jul 1988 |
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5155033 |
Estell et al. |
Oct 1992 |
|
Foreign Referenced Citations (1)
Number |
Date |
Country |
WO9202615 |
Feb 1992 |
WOX |
Non-Patent Literature Citations (3)
Entry |
Caldwell, R., et al., "Analysis of the Catalytic Importance of a Helix Dipole in Subtilisin Using Site-Directed Mutagenesis", American Chemical Society Abstract MBTD-73, Sep. 10-15 1989. |
Carter, P., et al., "Dissecting the Catalytic Triad of a Serine Protease", Nature, 332(7):564-568 (1988). |
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Divisions (1)
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Number |
Date |
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Parent |
218873 |
Mar 1994 |
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Continuations (1)
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Number |
Date |
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Parent |
13445 |
Feb 1993 |
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Continuation in Parts (1)
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Number |
Date |
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Parent |
566026 |
Aug 1990 |
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