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 Mating Factor α pro-peptide variant comprising:
 an amino acid substitution region located at positions 38-42 of Mating Factor α having the sequence 
 
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDV 
                 
                     
                 
                   AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR, 
                 
             
                
                
                
                
               
            
           
         
         wherein the amino acid substitution region is represented by formula (I): 
       
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                 
                 
                 
               
                     
                   X 38 -X 39 -X 40 -X 41 -X 42   
                   (I) 
                 
             
                
               
            
             
                
               
            
           
         
         wherein 
         X 38  is selected from F, L or V, 
         X 39  is selected from L, I, V or M, 
         X 40  is selected from A, G or R, 
         X 41  is selected from S, Y, L, I, V or M, 
         X 42  is selected from Y, W, L, I, V or M; 
         or wherein 
         X 38  is selected from I or V, 
         X 39  is selected from L, I, V or M, 
         X 40  is selected from A, G, Y, F, W, R, K, L, I, V or M, 
         X 41  is selected from Y, F or W, and 
         X 42  is selected from L or I; 
         with the proviso that X 38 -X 42  is not VIGYL (SEQ ID NO: 3). 
       
     
     
         2 . The Mating Factor α pro-peptide variant according to  claim 1  wherein
 X 38  is V, 
 X 39  is selected from L, I, V or M, 
 X 40  is selected from G, R or K, 
 X 41  is Y, and 
 X 42  is selected from L or I. 
 
     
     
         3 . The Mating Factor α pro-peptide variant according to  claim 1 , wherein
 X 38  is V, 
 X 39  is selected from L, I, V or M, 
 X 40  is selected from G or R, 
 X 41  is Y, and 
 X 42  is L. 
 
     
     
         4 . The Mating Factor α pro-peptide variant according to  claim 3 , wherein X 40  is R. 
     
     
         5 . The Mating Factor α pro-peptide variant according to  claim 1 , wherein three of the amino acid residues in X 38 -X 39 -X 40 -X 41 -X 42  are identical to the corresponding amino acid residues in VIGYL (SEQ ID NO:3). 
     
     
         6 . A 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 (SEQ ID NO: 3) sequence at positions 38-42 to comprise the amino acid sequence of formula (I):   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                 
                 
                 
               
                     
                   X 38 -X 39 -X 40 -X 41 -X 42   
                   (I) 
                 
             
                
               
            
             
                
               
            
           
         
       
       wherein 
       X 38  is selected from F, L or V, 
       X 39  is selected from L, I, V or M, 
       X 40  is selected from G or R, 
       X 41  is selected from S, Y, L, I, V or M, 
       X 42  is selected from Y, W, L, I, V or M, 
       with the proviso that X 38 -X 42  is not VIGYL (SEQ ID NO:3), 
       or wherein 
       X 38  is selected from I or V, 
       X 39  is selected from L, I, V or M, 
       X 40  is A, G, Y, F, W, R, K, L, I, V or M, 
       X 41  is selected from Y, F or W, and 
       X 42  is selected from L or I; 
       with the proviso that X 38 -X 42  is not VIGYL (SEQ ID NO:3),
 optionally an extension peptide, and 
 a GLP-1 peptide, 
 
       wherein said Mating Factor α pro-peptide is amino acid residues 20-85 in the polypeptide: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLL FINTTIASIAAKEEGVSLDKR (SEQ ID NO:2), and said variant comprises from 1-15 amino acid substitutions, deletions and/or additions relative to said polypeptide. 
     
     
         7 . An expression vector comprising a DNA sequence encoding a polypeptide according to  claim 1 . 
     
     
         8 . A host cell comprising the expression vector according to  claim 7 . 
     
     
         9 . 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 (SEQ ID NO:3) sequence at positions 38-42 of Mating Factor α pro-peptide to comprise the amino acid sequence of formula (I): 
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 1) 
                 
                 
                 
                 
               
                     
                   X 38 -X 39 -X 40 -X 41 -X 42   
                   (I) 
                 
             
                
               
            
             
                
               
            
           
         
         wherein 
         X 38  is selected from F, L or V, 
         X 39  is selected from L, I, V or M, 
         X 40  is selected from A, G or R, 
         X 41  is selected from S, Y, L, I, V or M, 
         X 42  is selected from Y, W, L, V or M; 
         with the proviso that X 38 -X 42  is not VIGYL (SEQ ID NO:3); 
         or wherein 
         X 38  is selected from I or V, 
         X 39  is selected from L, I, V or M, 
         X 40  is selected from A, G, Y, F, W, R, K, L, I, V or M, 
         X 41  is selected from Y, F or W, and 
         X 42  is selected from L or I; 
         with the proviso that X 38 -X 42  is not VIGYL (SEQ ID NO:3); 
         wherein said Mating Factor α pro-peptide is amino acid residues 20-85 in the polypeptide: 
       
       
         
           
                 
               
                   (SEQ ID NO: 2) 
                 
                   MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDV 
                 
                     
                 
                   AVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR. 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         10 . The method according to  claim 9 , wherein X 40  is R. 
     
     
         11 . The method according to  claim 9 , 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). 
     
     
         12 . The method according to  claim 9 , wherein said yeast carries at least one genetic modification reducing its capacity for O-glycosylation. 
     
     
         13 . The method according to  claim 9 , wherein the PMT1 gene in said yeast is deleted. 
     
     
         14 . The method according to  claim 9 , wherein said polypeptide comprises GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K]. 
     
     
         15 . The method according to  claim 9 , wherein said polypeptide consists of GLP-1(9-37)[K34R] or GLP-1(9-37)[K34R,G37K]. 
     
     
         16 . The method according to  claim 9 , wherein said polypeptide has an N-terminal extension. 
     
     
         17 . The method according to  claim 9 , wherein three of the amino acid residues in X 38 -X 39 -X 40 -X 41 -X 42  are identical to the corresponding amino acid residues in VIGYL (SEQ ID NO:3).