title: [Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists].
abstract: To investigate the effect of GST-KGDX (glutathione S-transferase-Lys-Gly-Asp-X) fusion protein, GP IIb/IIIa receptor antagonist, on platelet function in vitro. The KGDX (Lys-Gly-Asp-X) gene was assembled from 2 synthetic oligonucleotides, 36 bp in length, using BamH I and Xho I restriction enzyme sites at the end of the gene for cloning into the expression vector pGEX4T-1. Expression of fusion protein was directed by the tac promoter. The Escherichia coli DH5a contained the plasmid pGEX-4T-1-KGDX was expressed by 37 degrees C heat induction. The fusion protein of KGDX with glutathione S-transferase (GST-KGDX) was purified in one step from the bacterial lysate by glutathione-agarose beads for affinity chromatography. GST-KGDX was found to be soluble and abundant, the yield of 35 mg/L of cultures was obtained. The GST-KGDX was expressed in E. coli to a level of 48.02% of total cellular protein. GST-KGDX inhibited ADP-induced human platelet aggregation stronger than GST (P < 0.05 or < 0.01). In flow cytometry assay for fibrinogen binding, both GST and GST-KGDX inhibited platelet aggregation by binding with high affinity to GPIIb/IIIa. Mean fluorescence intensity of GST-KGDX fusion protein was significantly higher than that of GST. It is concluded that the GST-KGDX fusion protein can be produced by E. coli and used as an antiplatelet agent.
authors.affiliation: Department of Cardiovascular Disease, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China. happyzyp2002@sina.com
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Zha
authors.forename: Yong-Wen
authors.initials: YW
authors.lastname: Qin
authors.forename: Qing
authors.initials: Q
authors.lastname: Jing
authors.forename: Rui-Bin
authors.initials: RB
authors.lastname: Mu
mesh_terms: D000328:Adult; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D005982:Glutathione Transferase / Q000494:pharmacology; D006801:Humans; D008297:Male; D009842:Oligopeptides / Q000494:pharmacology; D010974:Platelet Aggregation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; C047981:arginyl-glycyl-aspartic acid; D005340:Fibrinogen; D005982:Glutathione Transferase