[Soluble expression, purification and characterization of Bm K IT in Escherichia coli by intein-mediated system].
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.
Xu
Cheng-Gang
CG
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
Fan
Xiao-Jun
XJ
Zhang
Zhi-Yun
ZY
Fu
Yue-Jun
YJ
Liang
Ai-Hua
AH
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
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