Chemically and thermodynamically stable insulin analogues and improved methods for their production

Abstract

The subject matter of this invention is directed towards chemically and thermodynamically stable single-chain insulin (SCI) analogues that are resistant to deamidation and fibrillation. The invention further discloses improved methods for the recombinant expression, purification and refolding of SCI.

What is claimed is: 
     
         1 . A single chain insulin (SCI) compound of the formula:
   Stable B chain-C′-Stable A chain  Formula (I)
   
       having the properties of high affinity for the insulin receptor and low affinity for the IGF-1 receptor with chemical and thermodynamic degradation profiles such that the SCI can be formulated and stored for extended periods of time without refrigeration;
 wherein Stable B chain and Stable A chain are modified human insulin chains; and 
 wherein C′ covalently links Stable B chain and Stable A chain, and is a peptide of about 5 to 9 amino acids comprising the following sequence: Y-P-G-D-X (SEQ ID NO: 1); wherein X is any amino acid. 
 
     
     
         2 . The SCI according to  claim 1 , wherein Stable B chain and Stable A chain are human insulin chains with chemical degradation resistance mutations. 
     
     
         3 . A polynucleotide comprising a nucleic acid sequence that encodes the single chain insulin compound according to  claim 1 . 
     
     
         4 . A recombinant vector comprising the polynucleotide according to  claim 3 . 
     
     
         5 . The recombinant vector according to  claim 4 , wherein said vector is a plasmid. 
     
     
         6 . The recombinant vector according to  claim 4 , comprising an inducible promoter. 
     
     
         7 . The recombinant vector according to  claim 6 , wherein said inducible promoter is IPTG. 
     
     
         8 . The recombinant vector according to  claim 4 , comprising a fusion tag. 
     
     
         9 . The recombinant vector according to  claim 8 , wherein the fusion tag is derived from oleosin. 
     
     
         10 . The recombinant vector accordingly to  claim 8 , wherein said fusion tag encodes a chemically cleavable sequence. 
     
     
         11 . A cell line transformed with the vector according to  claim 4 . 
     
     
         12 . A method for treating diabetes in a patient in need thereof comprising administering the single chain insulin compound according to  claim 1  to said patient. 
     
     
         13 . The method of  claim 12 , wherein the diabetes is type I diabetes. 
     
     
         14 . A method for producing single chain insulin comprising introducing a recombinant vector according to  claim 4  into an expression system, expressing a protein comprising a fusion tag and single chain insulin, cleaving said fusion tag from said single chain insulin, and isolating said single chain insulin. 
     
     
         15 . A method for obtaining purified single chain insulin (SCI) comprising producing said SCI according to  claim 14 , further comprising the step of affinity chromatography.