Expression and purification of the BmK Mm2 neurotoxin from the scorpion Buthus martensii Karsch and its biological activity test.
The gene encoding neurotoxin (BmK Mm2) from the scorpion Buthus martensii Karsch was expressed in Escherichia coli BL21 (DE3) at a level of 1.6 mg/L using expression plasmid pExSecI system. SDS-PAGE analysis of the cell lysate confirmed that gene BmK Mm2 was expressed in soluble form and the expressed production was secreted into Luria-Bertani (LB) culture medium from Escherichia coli. According to the characters of pExSecI expression system, the IgG binding domain-ZZ of Protein A is fused to the N-terminal of BmK Mm2. Recombinant BmK Mm2 (ZZ-BmK Mm2, pI 6.81, 22.007 kDa) was purified rapidly and efficiently by IgG-Sepharose 6 Fast Flow and Superdex-75 gel filtration chromatography, produced a single band on SDS-PAGE. Western blot analysis demonstrated that this protein was recombinant BmK Mm2. The results of MTT assay, morphological observation of nucleus and single cell gel electrophoresis showed that the expressed recombinant BmK Mm2 was toxic for glial cells of mice, which indicate that it has biological activity.
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
Yue-Jun
YJ
Fu
Bao-Feng
BF
Chai
Wei
W
Wang
Hui
H
Zhi
Li-Tian
LT
Yin
Ai-Hua
AH
Liang
D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D020161:Physical Chromosome Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012604:Scorpion Venoms / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D003470:Culture Media; D011994:Recombinant Proteins; D012604:Scorpion Venoms; C405242:scorpion neurotoxin BmK M2