Method for universal enzymatic production of bioactive peptides

Abstract

The invention provides methods for making peptides from a polypeptide containing at least one copy of the peptide using clostripain to excise the peptide from the polypeptide. The methods enable the use of a single, highly efficient enzymatic cleavage to produce any desired peptide sequence.

1 - 2 . (canceled) 
     
     
         3 . A method for producing a desired peptide having a C-terminal acidic, aliphatic or aromatic amino acid from a polypeptide, comprising:
 combining clostripain and the polypeptide comprising formula (III):
   (Linker-Xaa 3 -Peptide 1 ) n -Linker-Xaa 3 -Peptide 1   (III);
 
   wherein
 the desired peptide comprises Xaa 3 -Peptide 1 ; 
 Peptide 1  is any amino acid sequence other than Xaa 1 -Xaa 2 ; 
 n is an integer ranging from 0 to 50; 
 Xaa 3  is not an acidic amino acid; 
 Linker is a cleavable peptide linker having Formula (IV):
   (Peptide 5 ) m —Xaa 1 -Xaa 2   (IV);
 
 
   wherein
 m is an integer ranging from 0 to 50; 
 Xaa 1  is aspartic acid, glycine, proline or glutamic acid; 
 Xaa 2  is arginine; and 
 Peptide 5  is any single amino acid residue or amino acid sequence not containing Xaa 1 -Xaa 2 . 
   
     
     
         4 . The method of claim  1 , wherein the polypeptide is a soluble polypeptide. 
     
     
         5 . The method of claim  1 , wherein the cleavage is performed at about 18° C. to about 25° C. 
     
     
         6 . The method of claim  1 , wherein the cleavage is performed between a pH of about 5 to about 11. 
     
     
         7 . The method of claim  1 , wherein the concentration of clostripain is about 0.01 to about 3.0 units of clostripain per about 2 to about 5 mg polypeptide. 
     
     
         8 . The method of claim  1 , wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl 2 . 
     
     
         9 . The method of  claim 3 , wherein the Linker comprises Pro-Gly-Xaa 1 -Xaa 2 ; and Xaa 1  is aspartic acid. 
     
     
         10 . The method of  claim 3 , wherein the Linker comprises Val-Asp-Xaa 1 -Xaa 2 ; and Xaa 1  is aspartic. 
     
     
         11 . The method of  claim 3 , wherein the Linker comprises Ile-Thr-Xaa 1 -Xaa 2 ; and Xaa 1  is aspartic acid. 
     
     
         12 . The method of  claim 3 , wherein Peptide 5  is selected from the group consisting of Gly-Ser-, Cys-His-, Cys-His-Xaa-Xaa- and Val-Asp-; Xaa 1  is Aspartic acid or Glutamic acid; Xaa independently is any amino acid residue other than Arginine and m is an integer of 1 to 10. 
     
     
         13 . The method of claim  1 , wherein the desired peptide comprises anyone of SEQ ID NO:1-16. 
     
     
         14 . The method of claim  1 , wherein the polypeptide comprises a soluble four copy or six copy GLP-2(1-34) sequence. 
     
     
         15 - 71 . (canceled)