title: High-level expression of a functional human fibrinogen gamma chain in Escherichia coli.
abstract: Human fibrinogen gamma chain has been expressed intact at high levels in Escherichia coli. The construction of the expression plasmid, p253, is described. Synthesis of the recombinant protein is isopropyl-beta-D-thiogalactopyranoside-dependent and is driven by the tac promoter. Western analysis of E. coli lysates demonstrates a novel protein of approx. 45 kDa which cross-reacts with antisera to human fibrinogen gamma chains. The protein is not soluble in common E. coli lysis buffers and becomes soluble in 6 M guanidine.HCl or 6 M urea. Initial insolubility is due to interchain disulfide bond formation and to noncovalent interactions. Induced cells examined by phase-contrast microscopy contain dense inclusion bodies. A known function of the gamma chains of human fibrinogen is the clumping of Staphylococcus aureus Newman D2C cells [Hawiger et al., Biochemistry (1982) 1407-1413]. We demonstrate that suspensions of recombinant gamma chains retain the ability to clump cells from this strain of S. aureus.
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D003001:Cloning, Molecular; D003030:Coagulase; D004220:Disulfides; D004586:Electrophoresis; D004926:Escherichia coli / Q000235:genetics*; D005340:Fibrinogen / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006031:Glycosylation; D010084:Oxidation-Reduction; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003030:Coagulase; D004220:Disulfides; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D005340:Fibrinogen