Coordinated coexpression of thrombin

Abstract

The present invention provides methods of producing thrombin using coordinated coexpression systems, and particularly inducible coexpression systems, capable of controlled induction of expression of each gene product required for the production of thrombin.

What is claimed is: 
     
         1 . A method of producing a thrombin polypeptide, the method comprising:
 growing a culture of a host cell, wherein the host cell comprises at least one expression construct comprising a first inducible promoter selected from the group consisting of an  Escherichia  coli sugar-inducible promoter and a propionate-inducible promoter, and at least one polynucleotide sequence encoding a first thrombin polypeptide to be transcribed from said first inducible promoter,   wherein the host cell comprises at least one expression construct comprising a second inducible promoter, selected from the group consisting of an  Escherichia coli  sugar-inducible promoter and a propionate-inducible promoter, that is inducible by at least one inducer that is different than that of said first inducible promoter,   wherein none of said inducible promoters is a lactose-inducible promoter, and   wherein the host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one said inducible promoter; and   adding an inducer of said first inducible promoter to the culture.   
     
     
         2 . The method of  claim 1  wherein the host cell is a prokaryotic cell. 
     
     
         3 . The method of  claim 2  wherein the host cell is an  Escherichia coli  cell. 
     
     
         4 . The method of  claim 1  wherein the expression construct comprising said first inducible promoter is an extrachromosomal expression construct. 
     
     
         5 . The method of  claim 1  wherein the expression construct comprising said first inducible promoter, and the expression construct comprising said second inducible promoter, are comprised by one extrachromosomal polynucleotide. 
     
     
         6 . The method of  claim 1  wherein the first promoter is an  Escherichia coli  sugar-inducible promoter selected from the group consisting of the araBAD promoter, the rhaSR promoter, and the xylA promoter. 
     
     
         7 . The method of  claim 1  wherein at least one gene encoding a protein that metabolizes an inducer of at least one said inducible promoter is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB. 
     
     
         8 . The method of  claim 1  wherein the first thrombin polypeptide comprises the amino acid sequence of the thrombin A chain and the amino acid sequence of the thrombin B chain. 
     
     
         9 . The method of  claim 8  wherein the first thrombin polypeptide further comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of a SUMO tag, a 6×His amino acid sequence, and an ecarin cleavage site. 
     
     
         10 . The method of  claim 9  further comprising lysing the cultured host cells and contacting thrombin polypeptides present in the host cell lysate with a protease selected from the group consisting of SUMO protease and ecarin. 
     
     
         11 . The method of  claim 1  wherein said expression construct comprising the second inducible promoter further comprises at least one polynucleotide sequence encoding a second thrombin polypeptide to be transcribed from said second inducible promoter. 
     
     
         12 . The method of  claim 11  wherein at least one of said first thrombin polypeptide and said second thrombin polypeptide lacks a signal peptide. 
     
     
         13 . The method of  claim 11  wherein at least one of said first thrombin polypeptide and said second thrombin polypeptide comprises a human thrombin amino acid sequence variation selected from the group consisting of: E14eA/D141A/E18A; G14mP; R93A/W215A/E217A; T172Y/W215A/E217A; S195A; W215A; W215A/E217A; W215E; W215G; a deletion of residues E146-K149e; W215G/a deletion of residues E146-K149e; W215L; W215V; E217A; and E229K. 
     
     
         14 . The method of  claim 11  wherein said first thrombin polypeptide comprises the amino acid sequence of the thrombin A chain, and the second thrombin polypeptide comprises the amino acid sequence of the thrombin B chain. 
     
     
         15 . The method of  claim 11  wherein the second thrombin polypeptide further comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of a SUMO tag, a 6×His amino acid sequence, and an ecarin cleavage site. 
     
     
         16 . The method of  claim 15  further comprising lysing the cultured host cells and contacting thrombin polypeptides present in the host cell lysate with a protease selected from the group consisting of SUMO protease and ecarin. 
     
     
         17 . The method of  claim 1  further comprising lysing the cultured host cells and purifying thrombin polypeptides from the host cell lysate. 
     
     
         18 . The method of  claim 17  wherein said thrombin polypeptides are purified by contacting said thrombin polypeptides with at least one affinity medium comprising molecules selected from the group consisting of heparin, polyphosphate, fibrinogen, and thrombin-binding fragments of thrombomodulin. 
     
     
         19 . The method of  claim 17  wherein at least one of said thrombin polypeptides comprises at least one 6×His amino acid sequence, and said thrombin polypeptides are purified by contacting said thrombin polypeptides with an affinity medium comprising a metal selected from the group consisting of nickel and cobalt. 
     
     
         20 . The method of  claim 17  wherein said thrombin polypeptides purified from the host cell lysate have an activity selected from the group consisting of prothrombin-cleavage activity, fibrinogen-cleavage activity, fibrin-clotting activity, protein C-activation activity, chromogenic substrate S-2238-cleavage activity, and chromogenic substrate S-2366-cleavage activity.