Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli.
The CtxB gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that CtxB gene encodes 124 amino acid residues. The sequence of CtxB gene was almost the same as that of reported except for the codon of Thr 62. The expression plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid pGEX-4T-2, containing gst gene, immediately downstream of the T7 promoter. The expressed plasmid was introduced into E.coli BL21(DE3) cells and expression strain CTXB/BL21 was selected. SDS-PAGE analysis revealed that the GST-CTXB fusion protein was highly expression and accumulated up to 36% of bacterial soluble proteins after the induction by IPTG. A fusion protein of 40 kD was expressed as inclusion body. The fusion protein was refolded and purified. The purified fusion protein was cut by thrombin to obtain the purified CTXB protein.
He
Zhi-Yong
ZY
Shanghai Institute of Biochemistry, Chinese Academy of Science, Shanghai 200031, China. xfwu@sunm.shcnc.ac.cn
Li
Ming-Feng
MF
Zhang
Wei-Jie
WJ
Wu
Xiang-Fu
XF