[Construction,   expression and purification of trichosanthin mutant gene TCS(FYY163-165CSA);].
OBJECTIVE
To construct and express a trichosanthin (TCS) gene mutant and purify the expressed product in E.coli.


METHODS
The potential antigenic determinant  was predicted   on TCS molecule by computer modeling and induced for  site-directed mutation.  The  gene mutant TCS(FYY163-165CSA);  was amplified   by PCR using the genomic DNA of Trichosanthes kirilowii as a template and cloned into expression vector pRSET-A, then transfected into   E.coli BL21 (DE3) for expression by inducing with IPTG.  The expressed product  was identified  by Western blotting  and purified  by Ni-NTA affinity column chromatography.


RESULTS
The soluble target protein was successfully expressed in E.coli.  Homogenous TCS mutant protein was obtained after purification of expressed product.


CONCLUSIONS
The site-directed mutagenesis,  expression and purification of TCS provide a new approach for reconstructing TCS.
Department of Microbiology, Fourth Military Medical University, Xi'an, China.
Yuan
Y
Wang
You-hong
YH
Li
Zi-yu
ZY
Liu
Ying
Y
Shi
Zhi-kai
ZK
Xu
Jing
J
Xu
Zhi-dong
ZD
Li
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015978:Trichosanthin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D050505:Mutant Proteins; D015978:Trichosanthin