Cloning and expression of recombinant BmkNaTx12, a new voltage-gated sodium channel from scorpion Buthus martensii Karsch / 中国药科大学学报

ABSTRACT

A cDNA library was constructed from glands of wild Buthus martensii Karsch scorpion.A new sodium channel long-chain toxin BmkNaTx12 was identified by sequence alignment,and its full-length cDNA sequence was cloned.Sequence alignment showed that the sequence of BmkNaTx12 had high structural similarity with the β-toxin Cn12 from Mexican Centruroides noxius scorpion.The predicted structure of BmkNaTx12 was obtained by homologous model construction and the highest scoring model was selected.The protein structure was found to be stable at 300 ns by molecular dynamics optimization,and fluctuations at amino acids 20,35,and 52 are most like-ly due to the location of these amino acids in the loop region.Then,an expression system of recombinant BmkNaTx12-Fc fusion protein was constructed.The optimal expression time of recombinant protein was determined by Western blot and the fusion protein was successfully expressed in HEK293 cells.The purity of recombinant fusion protein which was obtained by protein A affinity chromatography was up to 95％ by SDS-PAGE gel electrophoresis and HPLC.In addition,the whole-cell patch-clamp assay results suggest that 1 μmoL/L BmkNaTx12-Fc can increase 20％ Nav1.7 peak current.These results laid a foundation for the further study of biological function.