Process for the production of alpha-human atrial natriuretic polypeptide

Abstract

The present invention relates to a process for the production of α-human atrial natriuretic polypeptide by recombinant DNA technology.

We claim:  
     
         1 . A process for the production of α-hANP by (1) culturing a microorganism transformed with an expression vector comprising a synthetic gene encoding an amino acid sequence of a protective peptide-fused α-hANP in a nutrient medium, (2) recovering the protective peptide-fused α-hANP from the cultured broth and (3) removing the protective peptide portion of the protective peptide-fused α-hANP.  
     
     
         2 . A process for the production of α-hANP of  claim 1 , in which the microorganism is bacteria.  
     
     
         3 . A process for the production of α-hANP of  claim 2 , in which the bacteria is a strain belonging to the genus Escherichia.  
     
     
         4 . A process for the production of α-hANP of  claim 3 , in which the strain is  Escherichia coli.    
     
     
         5 . A process for the production of α-hANP of  claim 1 , in which α-hANP gene portion of the synthetic gene is represented by the following DNA sequence:  
       
         
           
                 
                 
               
                     
                 
                   Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGT 
                     
                 
                   Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA 
                 
                     
                 
                   GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG 
                 
                   CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC 
                 
                     
                 
                   GGC TGT AAC TCT TTC CGT TAC-3′ 
                 
                   CCG ACA TTG AGA AAG GCA ATG-5′ 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         6 . A process for the production of α-hANP of  claim 1 , in which the protective peptide-fused α-hANP has the amino acid sequence represented in FIG. 17.  
     
     
         7 . A process for the production of α-hANP of  claim 1 , the protectice peptide portion of protective peptide-fused α-hANP is removed in the presence of API.  
     
     
         8 . A chemically synthsized gene encoding amino acid sequence of α-hANP: 
 H-Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH  
 
     
     
         9 . The chemically synthsized gene of  claim 8 , which is represented by the following DNA sequence:  
       
         
           
                 
                 
               
                     
                 
                   Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGT 
                     
                 
                   Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA 
                 
                     
                 
                   GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG 
                 
                   CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC 
                 
                     
                 
                   GGC TGT AAC TCT TTC CGT TAC-3′ 
                 
                   CCG ACA TTG AGA AAG GCA ATG-5′ 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         10 . A chemically synthesized protective peptide gene having DNA sequence encoding lysine as C-terminal of the protective peptide.  
     
     
         11 . The chemically synthesized protective peptide gene of  claim 10 , which is represented by the DNA sequence of FIG. 4.  
     
     
         12 . A recombinant vector comprising chemically synthesized α-hANP gene.  
     
     
         13 . A recombinant vector of  claim 13 , in which the α-hANP gene is represented by the following DNA sequence:  
       
         
           
                 
                 
               
                     
                 
                   Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGT 
                     
                 
                   Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA 
                 
                     
                 
                   GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG 
                 
                   CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC 
                 
                     
                 
                   GGC TGT AAC TCT TTC CGT TAC-3′ 
                 
                   CCG ACA TTG AGA AAG GCA ATG-5′ 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . A recombinant vector comprising chemically synthesized gene represented by the DNA sequence of FIG. 17.  
     
     
         15 . A recombinant vector comprising chemically synthesized gene represented by the DNA sequence of FIG. 4.  
     
     
         16 . A transformant comprising expression vector of chemically synthesized α-hANP gene.  
     
     
         17 . A transformant of  claim 16 , in which the α-hANP gene is presented by the following DNA sequence:  
       
         
           
                 
                 
               
                     
                 
                   Coding:    5′-TCT CTG CGT AGA TCC TCT TGC TTT GGT 
                     
                 
                   Noncoding: 3′-AGA GAC GCA TCT AGG AGA ACG AAA CCA 
                 
                     
                 
                   GGC CGT ATG GAC CGC ATC GGT GCT CAG TCC GGT CTG 
                 
                   CCG GCA TAC CTG GCG TAG CCA CGA GTC AGG CCA GAC 
                 
                     
                 
                   GGC TGT AAC TCT TTC CGT TAC-3′ 
                 
                   CCG ACA TTG AGA AAG GCA ATG-5′ 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         18 . A cleaving method of the fused-protein composed of a peptides having a lysine between a protective peptide and a targent peptide in the presence of API.  
     
     
         19 . Synthetic trp promoter III represented by the DNA sequence of FIG. 3.