Expression of anti-neuroexcitation peptide (ANEP) of scorpion Buthus martensii Karsch in Escherichia coli.
According to the cDNA sequence of anti-neuroexcitation peptide of scorpion Buthus martensii Karsch, the putative mature anti-neuroexcitation peptide (ANEP) encoding DNA fragment was obtained by a PCR method, then was cloned into expression plasmid pET28a, fused with His tag at its 3' end. When expressed in E. coli BL21 (DE3), the expression of recombinant ANEP was 15% of total cellular proteins, while most recombinant ANEP products existed in the form of insoluble inclusion bodies. Coexpression of molecular chaperones or protein disulfide isomerase could not improve its solubility. The recombinant ANEP in the cell lysate was purified to homogeneity by metal chelating affinity chromatography and Superdex 30 chromatography. In bioassay with convulsive mice model induced by thiosemicarbazide, recombinant ANEP could apparently delay the convulsion seizure of model animals by 18% and showed anti-neuroexcitatory activity.
Zhang
J H
JH
State Key Laboratory of Pharmaceutical Biotechnology, College of Life Sciences, Nanjing University, PR China.
Hua
Z C
ZC
Xu
Z
Z
Zheng
W J
WJ
Zhu
D X
DX
D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D008297:Male; D051379:Mice; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012604:Scorpion Venoms / Q000235:genetics* / Q000494:pharmacology; D012605:Scorpions / Q000737:chemistry* / Q000235:genetics; D012640:Seizures / Q000139:chemically induced / Q000517:prevention & control; D012663:Semicarbazides; D014162:Transfection
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D012663:Semicarbazides; C058792:anti-epilepsy peptide, Buthrus martensii; C005151:thiosemicarbazide; D012313:RNA