title: Modular design, expression and characterization of novel bifunctional mutants of fibrolase with combined platelet aggregation-inhibition and fibrinolytic activity.
abstract: Fibrolase is a non-hemorrhagic zinc metalloproteinase found in southern copperhead snake (Agkistrodon contortrix contortrix) venom that acts directly on fibrin clots and does not require plasminogen or any other blood-borne intermediate for activity. Chimeras of fibrolase with RGD peptides conferring antiplatelet activity have been synthesized covalently, but we describe a simpler, cheaper and less toxic method, using site-directed mutagensis. Fibrolase variants that constitute the arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD) motif were constructed using site-directed mutagenesis. Chimeric genes of fibrolase were expressed in Escherichia coli to obtain the bifunctional chimeric molecule of fibrolase that can inhibit platelet aggregation. After refolding and purification, platelet-targeted thrombolysis and antiplatelet aggregation of the target chimeric protein were determined. The mutant RGD-F2, using the GPRGDWRMLG peptide to replace the TSVSHD sequence between sites 69 and 72, not only had almost the same catalytic ability as wild-type fibrolase but also a strong ability to inhibit platelet aggregation.
authors.affiliation: Department of Bioengineering, Zhengzhou University, 450001, Zhengzhou, Henan, China.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Fang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.forename: San-Jun
authors.initials: SJ
authors.lastname: Chen
authors.forename: Zhen-Xia
authors.initials: ZX
authors.lastname: Bao
authors.forename: Yun-Fei
authors.initials: YF
authors.lastname: Qin
authors.forename: Zhao-Yu
authors.initials: ZY
authors.lastname: Zhu
authors.forename: Jin-Mei
authors.initials: JM
authors.lastname: Zhao
authors.forename: Jia
authors.initials: J
authors.lastname: Mai
authors.forename: Shou-Tao
authors.initials: ST
authors.lastname: Zhang
mesh_terms: D017836:Agkistrodon / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D005342:Fibrinolysis / Q000187:drug effects; D005343:Fibrinolytic Agents / Q000378:metabolism / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D008666:Metalloendopeptidases / Q000235:genetics* / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000235:genetics*; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D016415:Sequence Alignment
chemical_list: D005343:Fibrinolytic Agents; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; D008666:Metalloendopeptidases; C058789:fibrolase