title: [Purification and characterization of a kringle-deficit mutant of human plasminogen with Arg-Gly-Asp tripeptide expressed in Pichia pastorsis].
abstract: To obtain a recombinant human plasminogen (hPLG) with potential anti-platelet aggregation activity, we cloned the cDNA coding Pro544 to Asn791 of hPLG, a kringle-deficit derivative (hPLG-deltaK). The Pro559 in activation loop was then mutated into Asp559 to provide Arg-Gly-Asp (RGD) motif. The constructed pPICZalphaA-RGD-HPLG-deltaK plasmid was expressed in yeast Pichia pastoris GS115, which produced RGD-hPLG-deltaK about 0.160 g/L broth. After affinity chromatography, the purity of the recombinant protein reached above 90%. Western blotting test confirmed that it retained the immunological reaction capability as human PLG. Its urokinase activation rate in 24 hours and its fibrinolytic activity made no deference against native hPLG-deltaK (P=0.630, n=5). Importantly, after activation by urokinase, RGD-hPLG-deltaK showed a significantly higher platelet aggregation inhibition rate (Ri) (21.8% +/- 1.57%) than hPLG-deltaK (3.8% +/- 0.33%) (P=0.000, n=5). These results proved that we constructed an hPLG mutant with anti-platelet aggregation activity, which made a foundation for developing innovative thrombolytic drugs with multifunction.
authors.affiliation: Life Science and Bio-pharmaceutics College, Guangdong Pharmaceutical University, Guangzhou 510006, China.
authors.forename: Wu
authors.initials: W
authors.lastname: Chen
authors.forename: Maocai
authors.initials: M
authors.lastname: Wu
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Wu
authors.forename: Jianzhong
authors.initials: J
authors.lastname: Yang
authors.forename: Zhenlin
authors.initials: Z
authors.lastname: Chen
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Huang
authors.forename: Xinyong
authors.initials: X
authors.lastname: Zhang
authors.forename: Yun
authors.initials: Y
authors.lastname: Xiao
mesh_terms: D006801:Humans; D018082:Kringles / Q000235:genetics*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D010958:Plasminogen / Q000096:biosynthesis / Q000235:genetics*; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification / Q000494:pharmacology; D017354:Point Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D010958:Plasminogen