Plasmid for producing human insulin

Abstract

Recombinant plasmids are described that carry the structural gene for human proinsulin in expressible form. The human proinsulin structural gene is fused to a functional transport sequence. Host organisms transformed by the recombinant plasmids express a preproinsulin in vivo, and is transported across the host organism's cell wall. Following its recovery, in vitro methods are used to convert the human proinsulin to human insulin.

What is claimed is: 
     
       1. A plasmid capable of replication in an E. coli host organism, said plasmid comprising a DNA sequence of plasmid pJW2172 encoding the amino acid sequence of the ampicillinase leader peptide-human preproinsulin, said DNA sequence capable of expression in said E. coli host transformed with said plasmid to make preproinsulin product comprising a partial ampicillin leader sequence and partial human preproinsulin wherein the partial presequence thereof is perfectly fused to the partial aapicillin leader sequence, wherein said preproinsulin product, when processed in-vivo by an E. coli host is transported across the host cell membrane to form human proinsulin. 
     
     
       2. A plasmid according to claim 1 which is plasmid pJW2172. 
     
     
       3. An E. coli host organism transformed with a plasmid capable of replication in an E. coli host organism, said plasmid comprising a DNA sequence of plasmid pJW2172 encoding the amino acid sequence of the ampicillinase leader peptide-human preproinsulin, said DNA sequence capable of expression in said E. coli host transformed with said plasmid to make preproinsulin product comprising a partial ampicillin leader sequence and partial human preporinsulin wherein the partial presequence thereof is perfectly fused to the partial ampicillin leader sequence, wherein said preproinsulin product, when processed in vivo by an E. coli host is transported across the host cell membrane to form human proinsulin. 
     
     
       4. An E. coli host organism according to claim 3 which is capable of correctly processing the preproinsulin product in vivo to human proinsulin and transporting the human proinsulin across the hosts's cell membrane. 
     
     
       5. An E. coli host organism according to claim 4 which is E. coli strain K-12. 
     
     
       6. An E. coli host organism according to claim 5 transformed with plasmid pJW2172. 
     
     
       7. A preproinsulin product coaprising a polypeptide with the amino acid sequence encoded by the sequence of plasmid pJW2172, said polypeptide comprising a partial ampicillinase leader sequence and partial human preproinsulin, said partial presequence thereof perfectly fused to said partial ampicillinase leader sequence, wherein said preproinsulin product, when processed in vivo by an E. coli host is transported across the host cell membrane to form human proinsulin. 
     
     
       8. A process for producing human insulin comprising: (1) providing a plasmid capable of replication in an E. coli host organism, said plasmid comprising a DNA sequence of plasmid pJW2172 encoding the amino acid sequence of the ampicillinase leader preproinsulin, said DNA sequence capable of expression in said E. coli host transformed with said plasmid to make preproinsulin product comprising a partial ampicillin leader sequence and partial human preproinsulin wherein the partial presequence thereof is perfectly fused to the partial ampicillin leader sequence, wherein said preproinsulin product, when processed in vivo by an E. coli host is transported across the host cell membrane to form human proinsulin;   (2) transforming, with the plasmid of step (1), an E. coli host organism wh1ch is capable of correctly processing the preproinsulin product in vivo to human proinsulin and transporting the human proinsulin across the host's cell membrane;   (3) growing the transformed host organism of step (2) under conditions e for expression of the gene comprising the structural gene which codes for the amino acid sequence encoded by the ampicillinase leader peptide-human preproinsulin-coding DNA sequence of plasmid pJW2172;   (4) allowing the transformed host organism of step (2) to process in vivo the preproinsulin product, expressed from the gene comprising the structural gene which codes for the amino acid sequence encoded by the ampicillinase leader peptide-human preproinsulin-coding DNA sequence of plasmid pJW2172 during the growth of step (3), to human proinsulin and accumulate said human proinsulin outside the host's cell membrane;   (5) recovering the human proinsulin accumulated during the process of step (4);   (6) converting in vitro the human proinsulin recovered in step (5) into human 1nsulin; and   (7) purifying the human insulin produced in step (6).   
     
     
       9. A process according to claim 8 wherein the E. coli host organism is of E. coli strain K-12. 
     
     
       10. A process according to claim 9 wherein the plasmid is pJW2172.