C-terminal modification of polypeptides

Abstract

The invention relates to a mutated trypsin comprising an amino acid substitution both at position K60 and D189, and at least one more amino acid substitution by histidine at position N143 or position E151. Such trypsin mutant has a preferred cleavage site comprising the amino acids Xaa 1 -Xaa 2 -His, wherein Xaa 1 is L, Y or F and Xaa 2 is R or K. The invention also relates to a man-made polypeptide comprising a target peptide and the above cleavage site as well as to a method of producing C-terminally modified target peptides by using this mutated trypsin.

1 . A mutated trypsin comprising an amino acid substitution at position K60 and at position D189, and an amino acid substitution by histidine at position N143 or position E151.  
     
     
         2 . The mutated trypsin of  claim 1  wherein K60 is substituted by E or D.  
     
     
         3 . The mutated trypsin of  claim 1  wherein D189 is substituted by K, H or R.  
     
     
         4 . Use of a polypeptide comprising a target peptide and a restriction site peptide comprising the cleavage site Xaa 1 -Xaa 2 -His, wherein Xaa 1  is L, Y or F, and Xaa 2  is R or K, wherein said restriction site peptide overlaps with the target peptide by the amino acid Xaa 1  at the C-terminal end of said target peptide as a substrate of a trypsin mutant according to any of  claims 1  to  3 .  
     
     
         5 . A method of producing a C-terminally transacylated target peptide comprising the steps of: 
 providing a polypeptide comprising a target peptide and a restriction site peptide comprising the cleavage site Xaa 1 -Xaa 2 -His, wherein Xaa 1  is L, Y or F, and Xaa 2  is R or K, wherein said restriction site peptide overlaps with the target peptide by the amino acid Xaa 1  at the C-terminal end of said target peptide,    bringing said peptide into contact with a trypsin mutant according to any of  claims 1  to  3  under conditions allowing for endoproteolytic cleavage after Xaa 1  and formation of an endoprotease target peptide peptide-acyl-intermediate,    adding an appropriate nucleophile, and,    upon nucleophilic attack and binding of said nucleophile to the C-terminus of the target peptide, releasing the mutated trypsin from the endoprotease target peptide-acyl-intermediate.    
     
     
         6 . The method of  claim 5  wherein said nucleophile is selected from the group consisting of primary amines, imines, secondary amines, thiol and hydroxyl.  
     
     
         7 . The method of  claim 5  wherein said nucleophile comprising modification is selected from the group consisting of an amino acid amide, a peptide, a peptide amide, a label, a labeled amino acid amide, a labeled peptide, a labeled peptide amide, and polyethyleneglycol.  
     
     
         8 . The method of  claim 7  wherein said modification is polyethyleneglycol.  
     
     
         9 . A nucleotide sequence coding for the mutated trypsin as defined in any of  claims 1  to  3 .