title: Expression, purification and characterization of Gloydius shedaoensis venom gloshedobin as Hsp70 fusion protein in Pichia pastoris.
abstract: Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis was expressed as Hsp70 fusion protein from the construct pPIC9K/hsp70-TLE in the yeast Pichia pastoris. By fusing gloshedobin to the C-terminus of Hsp70, an expression level of 44.5mg Hsp70-gloshedobin per liter of culture was achieved by methanol induction. The fusion protein secreted in the culture medium was conveniently purified by two chromatographic steps: Q-Sepharose FF and Superdex 200. The purified enzyme had an apparent molecular mass of 98 kDa according to SDS-PAGE analysis, and exhibited fibrinogenolytic activity that preferentially degraded fibrinogen alpha-chain. The enzyme also degraded fibrinogen beta-chain to a lesser extent, while showing no degradation toward the gamma-chain. A fibrinogen clotting activity of 499.8 U/mg was achieved by the enzyme, which is within the range reported for other thrombin-like enzymes. Hsp70-gloshedobin had strong esterase activity toward the chromogenic substrate N alpha-p-tosyl-Gly-Pro-Arg-p-nitroanilide, and this activity was optimal at pH 7.5 and 50 degrees C, and was completely inhibited by PMSF, but not by EDTA. We concluded that Hsp70 has no effect on the physiochemical and biochemical properties of gloshedobin. Although applying a fusion partner with very big molecular weight is unusual, Hsp70 proved its advantage in soluble expression of gloshedobin without affecting its fibrinogenolytic activity. And this positive result may provide an alternative strategy for the expression of thrombin-like enzymes in microbial system.
authors.affiliation: Department of Bioscience and Biotechnology, Dalian University of Technology, No. 2 Linggong Road, Dalian 116024, China.
authors.forename: Daping
authors.initials: D
authors.lastname: Yang
authors.forename: Mingli
authors.initials: M
authors.lastname: Peng
authors.forename: Hua
authors.initials: H
authors.lastname: Yang
authors.forename: Qing
authors.initials: Q
authors.lastname: Yang
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Xu
mesh_terms: D000818:Animals; D002847:Chromatography, Agarose; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009842:Oligopeptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013917:Thrombin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014757:Viper Venoms / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017819:Viperidae / Q000235:genetics*
chemical_list: D018840:HSP70 Heat-Shock Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014757:Viper Venoms; C033785:N(alpha)-4-tosyl-glycyl-prolyl-arginine-4-nitroanilide; D005340:Fibrinogen; D013917:Thrombin