Insulin derivatives with a terminal lpxt motif

Abstract

The present invention relates to insulin derivatives which, in comparison to insulin glulisine and similar derivatives, has a simplified process for synthesis. In particular, the present invention relates to insulin derivatives or physiologically tolerable salts thereof in which the motif Leucine-Proline-X-Threonine appears at the end of the B-chain, where X is any amino acid residue. Due to the nature of the process, the A-chain and B-chain must begin with a Glycine amino acid residue.

1 . An insulin derivative or a physiologically tolerable salt thereof, in which the end of the B-chain is Leucine-Proline-X-Threonine, where X represents any amino acid residue, and in which both the A-chain and B-chain begin with a Glycine amino acid residue. 
     
     
         2 . A process of expressing a fully folded functional two chain insulin derivate that requires no further processing to make it functionally active, said process comprising steps of
 i) cloning an insulin derivative peptide and sortase in a yeast, such as  Pichia pastoris , or a bacteria, such as  Escherichia coli ; wherein, the sequence coding for sortase is put under the control of a constitutive or inducible promoter,   ii) co-expressing the said peptide and sortase; and   iii) obtaining a fully functional insulin derivative with a Leucine-Proline-X-Threonine motif at the terminal of the B chain, where X is any amino acid residue.   
     
     
         3 . The process as claimed in  claim 2 , wherein the gene encoding the single chain insulin derivative peptide is set forth as SEQ ID No. 1, or the expressed peptide contains SEQ ID No. 2 starting from the fourth amino acid residue. 
     
     
         4 . The process as claimed in  claim 2 , wherein the sortase gene was derived from bacteria of the order Lactobacillales or an organism genetically modified to contain the genetic sequence of a Lactobacillales bacteria. 
     
     
         5 . The process as claimed in  claim 2 , wherein sortase is expressed extracellularly by one host organism, and the single chain peptide is expressed separately by another host organism of the same species, or of a different species. 
     
     
         6 . A process of converting a single chain peptide into fully folded biologically active insulin derivative, said method comprising steps of:
 i) obtaining a host cell containing a nucleotide sequence encoding a single chain peptide precursor of an insulin derivative;   ii) combining the single chain peptide with sortase to convert the insulin derivative precursor into a fully folded, biologically active insulin derivative.   
     
     
         7 . An insulin derivative or a physiologically tolerable salt thereof, having the formula I, prepared in accordance with the process of  claim 2 : 
       
         
           
           
               
               
           
         
       
       in which
 (A1-A5) are the amino acid residues in the positions A1 to A5 of the A chain of human insulin or animal insulin, 
 (A12-A17) are the amino acid residues in the positions A12 to A17 of the A chain of human insulin or animal insulin, 
 A8 and A10 are the amino acid residues in positions A8 and A10 of the A chain of human insulin or animal insulin, 
 A9 is a serine residue (Ser) or alanine residue (Ala), 
 A19 is Tyr, Phe or Ser, 
 A21 is Asn, Asp, Gly, Ser, Thr or Ala, 
 (B4-B6) are the amino acid residues in the positions B4 to B6 of the B chain of human insulin or animal insulin, 
 (B8-B18) are the amino acid residues in the positions B8 to B18 of the B chain of human insulin or animal insulin, 
 (B23-B26) are the amino acid residues in the positions B23 to B26 of the B chain of human insulin or animal insulin, 
 B3 is Arg, Lys, His, Ala or Asn, 
 B20 is a glycine residue (Gly) or alanine residue (Ala), 
 B21 is a glutamic acid residue (Glu) or alanine residue (Ala), 
 B22 is an arginine residue (Arg) or alanine residue (Ala), 
 B29 is any naturally occurring amino acid residue.