Expression and purification of recombinant glycoprotein (GP) IIb/IIIa receptor antagonists.
- Author:
Yan-Ping ZHA
1
;
Yong-Wen QIN
;
Qing JING
;
Rui-Bin MU
Author Information
1. Department of Cardiovascular Disease, Changhai Hospital, The Second Military Medical University, Shanghai 200433, China. happyzyp2002@sina.com
- Publication Type:Journal Article
- MeSH:
Adult;
Escherichia coli;
genetics;
Female;
Fibrinogen;
metabolism;
Flow Cytometry;
Glutathione Transferase;
pharmacology;
Humans;
Male;
Oligopeptides;
pharmacology;
Platelet Aggregation;
drug effects;
Platelet Aggregation Inhibitors;
isolation & purification;
pharmacology;
Platelet Glycoprotein GPIIb-IIIa Complex;
antagonists & inhibitors;
metabolism;
Recombinant Fusion Proteins;
biosynthesis;
isolation & purification;
pharmacology
- From:
Journal of Experimental Hematology
2002;10(6):535-539
- CountryChina
- Language:Chinese
-
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.
