title: Expression of a cloned human fibrinogen cDNA in Escherichia coli: synthesis of an A alpha polypeptide.
abstract: The construction of a plasmid, p166.9, for the controlled synthesis of the A alpha-chain of human fibrinogen in Escherichia coli is described. The plasmid combines the tac promoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from an E. coli plasmid beta-lactamase gene and a cDNA of the A alpha-chain of human fibrinogen. The tac promoter is repressed in lacIQ strains of E. coli and induced by isopropylthio-beta-D-galactoside (IPTG). Protein blot analysis of lysates of cells carrying p166.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A alpha-chain of human fibrinogen. The largest and predominant species corresponds to an apparent molecular weight of 63,000. When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A alpha-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA. Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A alpha, this indicates that the A alpha-peptide concentration in the culture media is 13 micrograms/ml.
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D013917:Thrombin / Q000378:metabolism
chemical_list: D046911:Macromolecular Substances; D005340:Fibrinogen; D013917:Thrombin