Purification, Gene Synthesis And Expression Of Trigramin, A Disintegrin From Trimeresurus Stejnegeri Venom

Objective:1. To purify disintegrin from Trimeresurus stejnegeri venom and to investigate its biological activities.2. To investigate gene synthesis of the Trigramin from Trimeresurus stejnegeri venom and its expression in Escherichia coli.Methods:1. The disintegrin was purified by gel filtration chromatography and reverse-phase high performance liquid chromatography (RP-HPLC). The purity and molecular weight of the disintegrin was analyzed by HPLC and SDS-PAGE. The inhibitory effect of the disintegrin on ADP-induced platelet aggregation was analyzed by platelet aggregation assays.2. The four fragments of Trigramin gene were artificially synthesized according to its nucleic acid sequence. Slowly annealing PCR methods were conducted to obtain the full length of Trigramin gene. The artificial gene was cloned into prokaryotic expression vector pET-42a(+) at BamH I/Sac I site. The cloned insert was identified by PCR and sequence analysis. The recombinant expression plasmid was transformed into E. coli BL21(DE3) and GST-Trigramin fusion protein expression was induced with IPTG. The fusion protein was purified by GSTrap FF affinity chromatography. The expressed protein was identified by SDS-PAGE.Results:1. A disintegrin was purified from Trimeresurus stejnegeri venom by gelfiltration chromatography and RP-HPLC. It is identified a single component by HPLC and SDS-PAGE analysis. Its molecular weight is about 9.5kDa.2. The disintegrin inhibits ADP-induced platelet aggregation in a dose-dependent manner. The IC50 of the inhibition is about 2.42μg/ml (250nM).3. The Trigramin gene was synthesized successfully and inserted into the prokaryotic expression vector pET-42a(+). The recombinant expression plasmid pET-42a(+)-Trigramin was constructed.4. GST-Trigramin fusion protein was expressed in E. coli BL21(DE3) and its amounts were about 30% of total bacterial proteins.5. The fusion protein, GST-Trigramin, inhibits platelet aggregation induced by ADP. The IC50 of the inhibition is about 27μg/ml (670nM)。Conclution:1. A disintegrin was purified from Trimeresurus stejnegeri venom. It is a potent platelet aggregation inhibitor.2. The Trigramin gene was synthesized and expressed in E. coli BL21(DE3). The recombinant GST-Trigramin fusion protein was produced.