Recombinant DNA method for production of parathyroid hormone

Abstract

A recombinant DNA molecule coding for human parathyroid hormone operably linked to an inducible bacterial promoter, a leader growth hormone sequence, and a selective enzymatic cleavage site, expression of the molecule in bacteria, and methods for producing large quantities of human parathyroid hormone and its variants.

What is claimed is:  
     
       1. A recombinant DNA molecule comprising: 
       (a) an expression control region, said region in operable linkage with:  
       (b) a leader sequence coding for human growth hormone amino acids 1-138 operably linked to:  
       (c) a nucleotide sequence coding for a selective enzymatic cleavage site, said nucleotide sequence being linked to and preceding a genetic sequence coding for human parathyroid hormone (hPTH) or a polypeptide variant thereof having the capacity to bind to a receptor for hPTH.  
     
     
       2. The molecule of  claim 1  wherein said control region includes a bacterial, viral, fungal, or mammalian promoter. 
     
     
       3. The molecule of  claim 2  wherein said promoter is selected from the group consisting of the right promoter of bacteriophage λ (P L ), the left promoter of bacteriophage λ (P R ), the  E. coli trp  promoter, the  E. coli recA  promoter, the  E. coli lacZ  promoter, the  E. coli lacI  promoter, the  E. coli gal  promoter, the  B. subtilis  α-amylase promoter, the  B. subtilis  α-28-specific promoter, the promoters of the bacteriophages of  Bacillus , and the  Streptomyces  promoters. 
     
     
       4. The molecule of  claim 3  wherein said promoter is the  E. coli trp  promoter. 
     
     
       5. The molecule of  claim 1  wherein said selective enzymatic cleavage site is cleaved by Factor Xa. 
     
     
       6. The molecule of  claim 5  wherein said selective enzymatic cleavage site is Ile-Glu-Gly-Arg. 
     
     
       7. The molecule of  claim 1  wherein said selective enzymatic cleavage site is cleaved by thrombin. 
     
     
       8. The molecule of  claim 1  wherein said selective enzymatic cleavage site is Leu-Val-Pro-Arg. 
     
     
       9. The molecule of  claim 8  wherein said human parathyroid hormone is hPTH (1-84). 
     
     
       10. The molecule of  claim 8  wherein said polypeptide variant thereof having the capacity to bind to a receptor for hPTH is truncated at the N-terminus. 
     
     
       11. The molecule of  claim 10  wherein said polypeptide variant thereof having the capacity to bind to a receptor for hPTH is selected from the group consisting of hPTH(2-84), hPTH(3-84), hPTH(4-84), hPTH(5-84), hPTH(6-84), hPTH(7-84), and hPTH(8-84). 
     
     
       12. The recombinant DNA molecule of  claim 1  which is a plasmid. 
     
     
       13. The plasmid of  claim 12  which is pGFP-1. 
     
     
       14. A host cell containing the molecule of  claim 1 . 
     
     
       15. The cell of  claim 14  which is prokaryotic. 
     
     
       16. The cell of  claim 15  which is bacterial. 
     
     
       17. The cell of  claim 16  which is of the species  E. coli.    
     
     
       18. The cell of  claim 14  which is eukaryotic. 
     
     
       19. The cell of  claim 18  which is a yeast cell or a mammalian cell. 
     
     
       20. A method of producing human parathyroid hormone (hPTH) or a polypeptide variant thereof having the capacity to bind to a receptor for hPTH, substantially free of natural contaminants, comprising: 
       (a) providing the molecule of  claim 7  in a host;  
       (b) expressing said human parathyroid hormone or a polypeptide variant thereof having the capacity to bind to a receptor for hPTH thereof; and  
       (c) obtaining a fusion protein containing a human growth hormone, fused to a selective enzymatic cleavage site, fused to hPTH(1-84) or a polypeptide variant thereof having the capacity to bind to a receptor for hPTH; and  
       (d) cleaving said fusion protein of step (c) with an enzyme recognizing said selective enzymatic cleavage site.  
     
     
       21. The method of  claim 20  further comprising: 
       (f) purifying said hPTH(1-84) or said polypeptide variant thereof having the capacity to bind to a receptor for hPTH.  
     
     
       22. A fusion protein comprising: 
       (a) a human growth hormone 1-138 fused to  
       (b) a selective enzymatic cleavage site, fused to  
       (c) hPTH(1-84) or a polypeptide variant thereof having the capacity to bind to a receptor for hPTH.  
     
     
       23. The protein of  claim 22  wherein said selective enzymatic cleavage site is cleaved by Factor Xa. 
     
     
       24. The protein of  claim 22  wherein said selective enzymatic cleavage site is Ile-Glu-Gly-Arg. 
     
     
       25. The protein of  claim 22  wherein said selective enzymatic cleavage site is cleaved by thrombin. 
     
     
       26. The protein of  claim 22  wherein said selective enzymatic cleavage site is Leu-Val-Pro-Arg. 
     
     
       27. The protein of  claim 22  wherein said polypeptide variant thereof having the capacity to bind to a receptor for hPTH is selected from the group consisting of hPTH(2-84), hPTH(3-84), hPTH(4-84), hPTH(5-84, hPTH(6-84), hPTH(7-84), and hPTH(8-84). 
     
     
       28. The protein of  claim 27  wherein said polypeptide variant thereof having the capacity to bind to a receptor for hPTH is hPTH(3-84). 
     
     
       29. The method of  claim 21  wherein said hPTH(   1 - 84   )  is purified using an HPLC RPC -   18  system and is eluted with a linear  30 - 70   %  acetonitrile/H   2   O gradient.   
     
     
       30. Human parathyroid hormone substantially free of natural contaminants produced according to the method of  claim 29 .