Expression and purification of Huwentoxin-I in baculovirus system.
Huwentoxin-I (HWTX-I) is a novel neurotoxin isolated from the venom of Orinithoctonus huwena. Based on its biological activity, HWTX-I could be developed as a pain-killer for clinical purpose. Production of HWTX-I by the bacterium or yeast expression systems resulted in poor yields and the purified protein was proved to have lower biological activity than that of native one. So, for the first time, we introduced a new method to express HWTX-I gene in Sf9 cells using baculovirus expression system. Recombinant HWTX-I was recognized by Western blotting and then purified by nickel-chelating affinity chromatography under native conditions. Recombinant HWTX-I showed identical amino acid sequence as native form and exhibited similar effect on muscular transmission with that of native form. These results indicate that the baculovirus expression system and native purification strategy are viable ways to produce active HWTX-I.
Ji
Wenjie
W
National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
Zhang
Xinyue
X
Hu
Huicong
H
Chen
Jiyuan
J
Gao
Yin
Y
Liang
Songping
S
An
Chengcai
C
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D005786:Gene Expression Regulation; D051379:Mice; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D030162:Reptilian Proteins; D013111:Spider Venoms / Q000235:genetics* / Q000302:isolation & purification* / Q000494:pharmacology; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D013997:Time Factors
D009498:Neurotoxins; D011994:Recombinant Proteins; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I