title: Expression of soluble and functional snake venom fibrinolytic enzyme fibrolase via the co-expression of DsbC in Escherichia coli.
abstract: Fibrolase is a non-hemorrhagic zinc metalloproteinase found in southern copperhead snake venom (Agkistrodon contortrix contortrix). It is capable of degrading fibrin clots that result from purified fibrinogen or blood plasma. The DNA of fibrolase was amplified by recursive PCR, and cloned into the pET25b(+) expression vector. The effect of co-expression of signalless versions of catalysts or molecular chaperones FkpA, Skp and DsbC in cytoplasm was examined. When co-expressed with DsbC, compared to the totally insoluble inclusion bodies of fibrolase expressed separately, more than 90 % of recombinant fibrolase was soluble, according to denaturing polyacrylamide gel electrophoresis analysis. We also determined that FkpA and Skp had no effects on the solubility of target protein when co-expressed with fibrolase in Escherichia coli. Fibrolase was successfully purified using metal ion affinity chromatography and hydrophobic chromatography, and a maximum yield of 20 mg/L fibrolase was obtained. Fibrinolytic activity of recombinant fibrolase was demonstrated using fibrin plate assays and fibrinogen hydrolysis.
authors.affiliation: Northwest A&F University, Key Laboratory of Agricultural Molecular Biology in Shaanxi Province, Yangling, Shaanxi Province, 712100, P.R. China.
authors.forename: Shou-Tao
authors.initials: ST
authors.lastname: Zhang
authors.forename: Jing
authors.initials: J
authors.lastname: Shi
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.forename: Ya-Fei
authors.initials: YF
authors.lastname: Qi
authors.forename: Ai-Guang
authors.initials: AG
authors.lastname: Guo
mesh_terms: D017836:Agkistrodon / Q000235:genetics*; D000818:Animals; D003435:Crotalid Venoms / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012995:Solubility
chemical_list: C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C058789:fibrolase; D019704:Protein Disulfide-Isomerases