Insulin production methods and pro-insulin constructs

Abstract

Novel pro-insulin having specific amino acid and/or nucleic acid modifications suitable for improved methods of insulin production are provided. Novel and highly efficient processes for preparing the pro-insulin preparations and preparations containing them are also disclosed. The novel pro-insulin preparations may be converted into human insulin useful in therapeutic preparations. Novel peptides of the C-peptide, and N terminus, including RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR, and MHHHHHHGGR respectively are provided, as well as the unique nucleic acid molecules encoding them.

1 .- 8 . (canceled) 
     
     
         9 . A process for preparing a composition enriched for an insulin analog employing a modified human pro-insulin peptide comprising:
 (a) preparing an isolated nucleic acid sequence encoding native human pro-insulin;   (b) modifying the isolated nucleic acid sequence by providing a molecular tag on the modified pro-insulin sequence so as to provide a histidine-tagged modified pro-insulin peptide;   (c) converting a lysine residue to an alanine residue at position 64 in a C-peptide region of the pro-insulin sequence to provide a modified pro-insulin derivative gene sequence;   (d) inserting the modified pro-insulin derivative gene sequence into a suitable vector to provide a vector comprising the modified pro-insulin derivative gene sequence;   (e) transfecting a culture of competent cells comprising  E. coli  cells with the vector to provide transformed  E. coli  cells;   (f) culturing the transformed  E. coli  cells under conditions suitable for expression of the modified pro-insulin derivative gene sequence;   (g) disrupting the population of transformed  E. coli  cells to provide a composition comprising inclusion bodies containing the modified human pro-insulin;   (h) solubilizing the composition to provide a composition comprising unfolded peptide;   (i) refolding the unfolded peptide to provide refolded human pro-insulin derivative peptide;   (j) passing the composition over a metal chelating column comprising nickel chelate to purify the composition and collecting a purified preparation of the refolded human pro-insulin derivative peptide;   (k) transforming the collected purified preparation of refolded human pro-insulin derivative peptide into Arg and Di-Arg peptide species by tryptic digestion;   (l) purifying the Arg and Di-Arg peptide species by passing the peptides through a reverse phase column; and   (m) transforming the purified Arg and Di-Arg peptide species into insulin by carboxypeptidase B digestion and harvesting an insulin analog.   
     
     
         10 . The process of  claim 9 , wherein the  E. coli  is a BL21 production  E. coli.    
     
     
         11 . The process of  claim 9 , wherein the peptide is further defined as comprising an amino acid sequence encoding a substituted human pro-insulin derivative protein having an amino acid sequence comprising: 
       
         
           
                 
               
                   MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVG 
                 
                     
                 
                   QVELGGGPGAGSLQPLALEGSLQARGIVEQCCTSICSLYQLENYCN. 
                 
             
                
                
                
               
            
           
         
       
     
     
         12 . The process of  claim 9 , wherein the reverse phase chromatography column is further defined as comprising a silica based media with a C4, C8, or C18 bonded phase. 
     
     
         13 . The process of  claim 9  wherein the suitable vector is a pTrcHis2A (Kan) vector. 
     
     
         14 . A pharmaceutical product comprising recombinant human insulin prepared by the process of  claim 9 . 
     
     
         15 . A process for preparing a human pro-insulin derivative comprising a C-peptide RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR, the process comprising:
 (a) preparing a pro-insulin peptide having a C-peptide amino acid sequence RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR;   (b) modifying the peptide to include a met-histidine-Gly-Gly-Arg tag at the N-terminus of the peptide;   (c) incorporating the nucleic acid sequence into an appropriate vector to provide a transformed vector; (pTrcHis vector);   (d) transforming a population of competent cells comprising  E. coli  cells with the vector to provide transformed  E. coli  cells;   (e) selecting transformed  E. coli  cells that express a peptide comprising an amino acid sequence -RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ-AR;   (f) culturing a composition of the selected transformed cells under conditions suitable for expression of the peptide having the amino acid sequence;   (g) solubilizing the composition comprising the cells; and   (h) purifying the human pro-insulin derivative from the culture.   
     
     
         16 . The process of  claim 15 , further comprising the step:
 (i) crystallizing the human pro-insulin derivative.   
     
     
         17 . The process of  claim 15 , further comprising preparing human insulin from the human pro-insulin derivative by enzymatic hydrolysis. 
     
     
         18 . The process of  claim 15  wherein the  E. coli  is a BL21 production  E. coli.    
     
     
         19 . A composition prepared by the process of  claim 17 , wherein the composition is essentially free of human pro-insulin.