Method for making acylated polypeptides

Abstract

The present invention related to a method of producing polypeptides in transformed host cells by expressing a precursor molecule of the desired polypeptide which are to be acylated in a subsequent in vitro step. The invention is also related to DNA-sequences, vectors and transformed host cells for use in the claimed method. Further, the present invention is related to certain precursors of the desired polypeptides and certain acylation methods. The invention provides a method for making polypeptides being preferentially acylated in certain lysine ε-amino groups.

What is claimed is:  
     
         1 . A method for making an acylated polypeptide, wherein said polypeptide comprises at least one lysine residue that is acylated on its ε-amino group, said method comprising: 
 (i) expressing in a suitable host cell a precursor molecule of the polypeptide, wherein said precursor comprises said polypeptide and an N-terminal extension, wherein said N-terminal extension is capable of protecting the desired polypeptide against proteolytic degradation and has a cleavage site different from Lys positioned at its C-terminal;  
 (ii) acylating the ε-amino group of at least one lysine residue in the desired polypeptide; and  
 (iii) removing the N-terminal extension by chemical and/or enzymatic cleavage.  
 
     
     
         2 . A method according to  claim 1 , wherein the acylating step is conducted after the removing step.  
     
     
         3 . A method according to  claim 2 , wherein the polypeptide is monoacylated.  
     
     
         4 . A method according to  claim 1 , wherein the N-terminal extension is up to 15 amino acid residues in length.  
     
     
         5 . A method according to  claim 4 , wherein the N-terminal extension is 3-12 amino acid residues in length.  
     
     
         6 . A method according to  claim 1 , wherein the polypeptide belongs to the GRF (growth hormone releasing factor) family of peptides having a His or Tyr in the N-terminal position and Ser, Ala or Gly in the next position.  
     
     
         7 . A method according to  claim 6 , wherein the polypeptide has a His-Ala, His-Gly, His-Ser or Tyr-Ala as the N-terminal sequence.  
     
     
         8 . A method according to  claim 1 , wherein the polypeptide is GLP-1 or GLP-2 or a GLP-1 or GLP-2 analogue.  
     
     
         9 . A method according to  claim 8 , wherein the polypeptide is Arg 34 GLP1 (7-37)  acylated in position Lys 26 .  
     
     
         10 . A method according to  claim 1 , wherein the cleavage site in the N-terminal extension is selected from the group consisting of Met, Asn, Pro, Gln, Cys and Arg-Arg.  
     
     
         11 . A method according to  claim 1 , wherein the N-terminal extension comprises a Glu-Glu sequence at the N-terminal end.  
     
     
         12 . A method according to  claim 1 , wherein the N-terminal extension has the sequence 
       X n  - - - X 1 —Y 
       wherein X n  - - - X 1  is a peptide sequence of from 1-14 amino acid residues in length, the sequence X n  - - - X 1 —Y having the function of a) protecting the expressed polypeptide from endoproteolytic cleavage, b) preventing acylation of the N-terminal end of the polypeptide and c) preventing precipitation caused by fibrillation during fermentation and downstream separation and purification steps; and Y is Met; Asn, Pro, Gln, Cys or Arg-Arg.  
     
     
         13 . A method according to  claim 12 , wherein X n  - - - X 1  is a peptide sequence of from 2-14 amino acid residues in length.  
     
     
         14 . A method according to  claim 12 , wherein X n  - - - X 1  is a peptide sequence of from 3-12 amino acid residues in length.  
     
     
         15 . A method according to  claim 1 , wherein the N-terminal extension is selected from the group consisting of Glu-Glu-Met; Glu-Glu-Ala-Glu-Met(SEQ ID NO:1); Glu-Glu-Ala-Glu-Asn(SEQ ID NO:2); Glu-Glu-Ala-Glu-Arg-Arg(SEQ ID NO:3); Gln; Glu-Pro-Gln(SEQ ID NO:4); Glu-Ala-Gln; Glu-Ala-Glu-Ala-Gln(SEQ ID NO:5); Glu-Ala-Glu-Ala-Glu-Ala-Gln(SEQ ID NO:6); Glu-Glu-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:7); Glu-His-Gly-Cys-Thr-Ser-Ile-Cys(SEQ ID NO:8); Glu-Glu-Ala-Arg-Met(SEQ ID NO:9); Glu-Glu-Arg-Asn(SEQ ID NO:10); Glu-Glu-Ala-Glu-Asn(SEQ ID NO:11); Glu-Glu-Arg-Ala-Arg-Arg(SEQ ID NO:12); Glu-Glu-Ala-Glu-Pro(SEQ ID NO:13); Glu-Glu-Gly-Glu-Pro(SEQ ID NO:14); Glu-Glu-Ala-Glu-Cys(SEQ ID NO:15); and Glu-Glu-Ile-Glu-Gly-Arg(SEQ ID NO:16).  
     
     
         16 . A method according to  claim 1 , wherein the host cell is a yeast cell.  
     
     
         17 . A method according to  claim 16 , wherein the yeast cell is a  Saccharomyces cerevisiae  cell.  
     
     
         18 . A precursor of a polypeptide, said precursor having the formula 
       N-terminal extension-Y-*polypeptide* 
       wherein Y is Met, Asn, Pro, Gin, Cys or Arg-Arg, the N-terminal extension has 1-14 amino acid residues and *polypeptide* is the remaining sequence of the polypeptide.  
     
     
         19 . A precursor according to  claim 18  having the formula 
       N-terminal extension-Y 1 —Y 2 - Y 3 - *polypeptide* 
       wherein Y 1  is Met, Asn, Pro, Gln, Cys or Arg-Arg; Y 2  is His or Tyr, Y 3  is Ala, Ser or Gly, the N-terminal extension has 1-14 amino acid residues and *polypeptide* is the remaining part of desired polypeptide.  
     
     
         20 . A precursor according to  claim 19 , wherein the polypeptide is GLP-1 or GLP-2 or a GLP-1 or GLP-2 analogue.  
     
     
         21 . A precursor according to  claim 20 , wherein the polypeptide is Arg 34 GLP1 (7-37)  acylated in position Lys 26 .