Gene Synthesis Of Cystatin From Snake Venom And Its Expression In E.coli.

Objective: To investigate gene synthesis of the cystatin from snake venom and its expression in Escherichia coli.Methods: The four fragments of cystatin gene of snake venom were artificially synthesized according to its amino acid sequence. Slowly annealing PCR methods were conducted to obtain the full length of cystatin 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-Cystatin fusion protein expression was induced with IPTG. The fusion protein was purified by GST·MagTM Agarose Beads. The expressed protein was identified by SDS-PAGE and Western-blot. The recombinant Cystatin protein was obtained by cleavage of GST-Cystatin fusion protein with Factor Xa and identified by Western-blot.Results: The cystatin gene from snake venom was successfully synthesized and inserted into the prokaryotic expression vector pET-42a(+). The recombinant expression plasmid pET-42a(+)-Cystatin was constructed . GST-Cystatin fusion protein was expressed in E.coli. BL21(DE3) and its amounts were about 30% of total bacterial proteins. The recombinant GST-Cystatin fusion protein from snake venom was purified by GST·MagTM Agarose Beads. The recombinant cystatin protein was obtained by cleavage of GST-Cystatin fusion protein with Factor Xa and has inhibitory activity against papain in primary assays. Conclution: The full length of cystatin gene from snake venom and its recombinant protein were obtained.