title: [Soluble expression, purification and characterization of Bm K IT in Escherichia coli by intein-mediated system].
abstract: To produce recombinant Buthus martensii Karsch insect toxin (BmK IT), BmK IT cDNA which fused a hexahistidine sequence at the C-terminus by PCR was inserted into pTWIN1 expression vector fused in frame with an upstream Ssp DnaB intein gene. The expression plasmid was transformed into E. coli BL21 (DE3) strain and protein expression was induced by IPTG. The CBD-Intein-BmK IT(his6) fusion protein was purified from cell lysates using Ni-NTA resin affinity chromatography. The intein was removed from fusion protein by on-column intein-mediated cleavage. BmK IT(his6) was purified through Superdex 75 gel chromatography to more than 95% homogeneity. The purified protein has both correct secondary structure and insecticidal activity.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
authors.forename: Cheng-Gang
authors.initials: CG
authors.lastname: Xu
authors.forename: Xiao-Jun
authors.initials: XJ
authors.lastname: Fan
authors.forename: Zhi-Yun
authors.initials: ZY
authors.lastname: Zhang
authors.forename: Yue-Jun
authors.initials: YJ
authors.lastname: Fu
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Liang
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D047668:Inteins / Q000235:genetics*; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D014170:Transformation, Genetic
chemical_list: C509305:Bmk IT toxin, Buthus martensii Karsch; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D012604:Scorpion Venoms; D006639:Histidine