A fusion protein of conotoxin MVIIA and thioredoxin expressed in Escherichia coli has significant analgesic activity.
omega-Conotoxin MVIIA (CTX MVIIA) is a potent and selective blocker of the N-type voltage-sensitive calcium channel in neurons. Its analgesic and neuroprotective effects may prove useful in treatment of severe pains and ischemia. In this paper, we report that a fusion form of CTX MVIIA with thioredoxin (Trx) has analgesic function. The DNA fragments were chemically synthesized and ligated to form the DNA sequence encoding CTX MVIIA. The synthetic gene was then cloned into the expression vector pET-32a(+) and the fusion protein Trx-CTX MVIIA containing 6x His-tag was purified by one-step metal chelated affinity chromatography (MCAC). The purity of final product was over 95% determined by HPLC and the yield of the fusion protein was approximately 40 mg/L. The analgesic function was detected by using mouse hot-plate assay. After intracranially administering fusion protein with the dose of 0.6 mg/kg, marked analgesia was observed. The analgesic effects (elevated pain thresholds) were dose-dependent and the biological half-life of the fusion toxin was approximately 1.6 h.
Department of Biochemistry, Zhejiang University Medical School, 353 Yan An Road, Hangzhou 310006, PR China. jzhan2k@cmm.zju.edu.cn
Jinbiao
J
Zhan
Xiaoying
X
Chen
Changmei
C
Wang
Jiwan
J
Qiu
Fengsen
F
Ma
Keyi
K
Wang
Shu
S
Zheng
D000700:Analgesics; D000818:Animals; D001522:Behavior, Animal / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017288:Pain Threshold / Q000187:drug effects*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology; D013879:Thioredoxins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*; D020960:omega-Conotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*
D000700:Analgesics; D011993:Recombinant Fusion Proteins; D020960:omega-Conotoxins; D013879:Thioredoxins; C078452:ziconotide