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| Expression, Purification and Identfication of Recombinant Conotoxin GeXIVAWT |
| GAO Bing-miao, LI Bao-zhu, WU Yong, LIN Bo, ZHU Xiao-peng, ZHANGSUN Dong-ting, LUO Su-lan |
| Key Lab for Tropical Biological Resources, Ministry of Education; Key Lab for Marine Drugs of Haikou, Haikou 570228, China |
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Abstract Conotoxin GeXIVAWT is a 28-amino acid peptide containing two pairs of disulfide bond isolated from the venom of worm-hunting Conus generalis Lonnaeus. Usually, the conotoxins produced by means of chemical synthesis was low yield, and it was difficult to be purified. A simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression was presented. The codons of novel conotoxin GeXIVAWT gene were optimized. Two pairs of primers were generated by chemical synthesis for construction of expression vectors. Recombinant expression vector pET22b(+)/pelB-GeXIVAWT fused with pelB signal peptide was successfully expressed in Escherichia coli. Recombinant pelB-GeXIVAWT without His-tag was purified by low concentrations of urea and ultrafiltration tube. Inactive inclusion bodies were transformed into active recombinant conotoxins by using dilution refolding method. It was further purified using HPLC and identified by mass spectrometry. Renaturing in vitro and biological activity experiment showed that the pelB-GeXIVAWT could inhibited the growth of Spodoptera frugiperda 9 (Sf-9) cell, as a foundation for the study of efficient insecticides.
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Received: 05 May 2012
Published: 25 September 2012
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