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 (SEQ ID NO:32), and MHHHHHHGGR (SEQ ID NO:36) respectively are provided, as well as the unique nucleic acid molecules encoding them.

1 . A nucleic acid sequence encoding a polypeptide comprising the sequence A-RREAEALQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO:32)-B, wherein said A chain and said B chain are native human insulin chains. 
     
     
         2 . A nucleic acid sequence encoding the polypeptide 
       
         
           
                 
                 
               
                   RREAEALQVGQVELGGGPGAGSLQPLALEGSLQA 
                   (SEQ ID NG:32) 
                 
                     
                 
                   R. 
                     
                 
             
                
                
                
               
            
           
         
       
     
     
         3 . An expression vector comprising the nucleic acid sequence of  claim 1 . 
     
     
         4 . The expression vector of  claim 3 , wherein the expression vector is Pro-insulin (His Tagged)/K64A/pTrcHis2A(Kan),His:tagged-Gly-Gly-Arg/Pro-insulin/(Kan). 
     
     
         5 . A microorganism transformed with the vector of  claim 4 . 
     
     
         6 . The microorganism of  claim 5  further defined as an  E. coli  transformed with plasmid Pro-Insulin (His Tagged)/K64A/pTcHis2A(Kan) plasmid. 
     
     
         7 . An amino acid sequence comprising 
       
         
           
                 
                 
               
                   RREAEALQVGQVELGGGPGAGSLQPLALEGSLQA 
                   (SEQ ID NG:32) 
                 
                     
                 
                   R. 
                     
                 
             
                
                
                
               
            
           
         
       
     
     
         8 . A composition enriched for a peptide encoded by the amino acid sequence of  claim 7 . 
     
     
         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 said nucleic acid sequence by providing a molecular tag on said 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 said 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 said modified pro-insulin derivative gene sequence;   (e) transfecting a culture of competent cells comprising  E. coli  cells with said 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 said population of transformed  E. coli  cells to provide a composition comprising inclusion bodies containing the modified human pro-insulin;   (h) solubilizing said composition to provide a composition comprising unfolded peptide;   (i) refolding said unfolded peptide to provide refolded human pro-insulin derivative peptide;   (j) passing said composition over a metal chelating column (Nickel chelate) to purify said composition and collecting a purified preparation of said 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 said Arg and Di-Arg peptide species by passing said 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: 
       
         
           
                 
               
                   (SEQ ID NO: 30) 
                 
                 
               
                   MHHHHHHGGRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVG 
                 
                     
                 
                   QVELGGGPGAGSLQPLALEGSLQARGIVEGCCTSICSLYGLENYCN. 
                 
             
                
               
            
             
                
                
                
               
            
           
         
       
     
     
         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 (SEQ ID NO:33), said process comprising:
 (a) preparing a pro-insulin peptide having a C-peptide amino acid sequence   
       
         
           
                 
                 
               
                   RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQA 
                   (SEQ ID NG:33) 
                 
                     
                 
                   R; 
                     
                 
             
                
                
                
               
            
           
         
         (b) modifying said peptide to include a met-histidine-Gly-Gly-Arg tag at the N-terminus of said peptide; 
         (c) incorporating said 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 said vector to provide transformed  E. coli  cells; 
         (e) selecting transformed  E. coli  cells that express a peptide comprising an amino acid sequence -RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQAR (SEQ ID NO:33); 
         (f) culturing a composition of said selected transformed cells under conditions suitable for expression of the peptide having said amino acid sequence; 
         (g) solubilizing said composition comprising said 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 said composition is essentially free of human pro-insulin.