Methods of use of serine protease variants having peptide ligase activity
AssigneeGenentech, Inc.
InventorsLars Abrahmsen, John Burnier, James A. Wells, David Y. Jackson
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Abstract
The invention relates to serine protease variants derived from precursor serine proteases via recombinant and/or chemical methods to form protease variants having improved peptide ligase activity. The invention also includes novel ligation substrates which in combination with the serine protease variants and a second ligation substrate are capable of forming a ligation product. The invention also relates to methods for forming such ligation products and the products formed thereby.
Claims27
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.
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.
Dependencies are inferred from the claim text.