Mating factor alpha pro-peptide variants

Abstract

The present invention is related to Mating Factor α pro-peptide variants useful for the recombinant expression of polypeptides comprising a GLP-1 peptide in yeasts. The invention is also related to DNA sequences, vectors and host cells for use in expressing polypeptides in yeasts.

1 . A method for recombinant expression of a polypeptide comprising a GLP-1 peptide in yeast comprising the culturing of a yeast strain comprising a DNA sequence encoding a processing and secretion signal upstream of the polypeptide, wherein said processing and secretion signal comprises a Mating Factor α pro-peptide variant having at least one substitution in the VIGYL sequence at positions 38-42 to comprise the amino acid sequence of the general formula (I):
   X 38 -X 39 -X 40 -X 41 -X 42  (SEQ ID NO:1)  (I)
 
 
       wherein
 X 38  is F, L, I or V; 
 X 39  L, I, V or M; 
 X 40  is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; 
 X 41  is S, Y, F, W, L, I, V or M; 
 X 42  is Y, W, L, I, V, M or S; 
 with the proviso that X 38 -X 42  is not VIGYS. 
 
     
     
         2 . The method according to  claim 1  wherein
 X 38  is F, L or V; 
 X 39  L, I, V or M; 
 X 40  is G or R; 
 X 41  is S, Y, L, I, V or M, and 
 X 42  is Y, W, L, V or M. 
 
     
     
         3 . The method according to  claim 1  wherein
 X 38  is I or V; 
 X 39  L, I, V or M; 
 X 40  is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; 
 X 41  is Y, F, or W, and 
 X 42  is L or I. 
 
     
     
         4 . The method according to  claim 1  wherein
 X 38  is V; 
 X 39  L, I, V or M; 
 X 40  is G or R; 
 X 41  is Y, and 
 X 42  is L. 
 
     
     
         5 . The method according to  claim 1 , wherein X 40  is R. 
     
     
         6 . The method according to  claim 1 , wherein said Mating Factor α pro-peptide variant has less than 10 amino acid residue changes outside of the X 38 -X 42  sequence as compared to the Mating Factor α pro-peptide as set out in SEQ ID NO:2 (amino acid residues 20-85). 
     
     
         7 . The method according to  claim 1 , wherein said yeast carries at least one genetic modification reducing its capacity for O-glycosylation. 
     
     
         8 . The method according to  claim 7 , wherein the PMT1 gene in said yeast is deleted. 
     
     
         9 . The method according to  claim 1 , wherein said polypeptide comprises GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K]. 
     
     
         10 . The method according to  claim 1 , wherein said polypeptide consists of GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K]. 
     
     
         11 . The method according to  claim 9 , wherein said polypeptide has an N-terminal extension. 
     
     
         12 . Mating Factor α pro-peptide variant having at least one substitution in the VIGYL sequence at positions 38-42 to comprise the amino acid sequence of the general formula (I):
   X 38 -X 39 -X 40 -X 41 -X 42  (SEQ ID NO:1)  (I)
 
 
       wherein
 X 38  is F, L, I or V; 
 X 39  L, I, V or M; 
 X 40  is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M; 
 X 41  is S, Y, F, W, L, I, V or M; 
 X 42  is Y, W, L, I, V, M or S; 
 
       with the proviso that X 38 -X 42  is not VIGYS, VIDYS, VATYL, VIGYR, or AIGYL. 
     
     
         13 . GLP-1 precursor which is a fusion polypeptide comprising:
 A pre-peptide,   A Mating Factor α pro-peptide variant having at least one substitution in the VIGYL sequence at positions 38-42 to comprise the amino acid sequence of the general formula (I):
   X 38 -X 39 -X 40 -X 41 -X 42  (SEQ ID NO:1)  (I)
 
   wherein   X 38  is F, L, I or V;   X 39  L, I, V or M;   X 40  is A, G, S, E, Q, Y, F, W, R, K, H, L, I, V or M;   X 41  is S, Y, F, W, L, I, V or M;   X 42  is Y, W, L, I, V, M or S;   with the proviso that X 38 -X 42  is not VIGYS,   Optionally an extension peptide, and   A GLP-1 peptide.   
     
     
         14 . Expression vector comprising a DNA sequence encoding the polypeptide according to  claim 12 . 
     
     
         15 . Host cell comprising the expression vector according to  claim 14 .