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 1.6mg/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, 22.007kDa) 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. [Copyright &y& Elsevier]