Wang Yuan, Shi Ying, Liu Zi-yu, Xu Zhi-kai, Xu Jing, Li Zhi-dong
Department of Microbiology, The Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2011 Apr;27(4):392-4.
To construct and express a trichosanthin(TCS)gene mutant and purify the expressed product.
Predict the potential antigenic determinant on TCS molecule by computer modeling and induce site-directed mutation. Amplify gene mutant TCS(RL28-29CG); by PCR using the genomic DNA of Trichosanthes kirilowii as a template and insert into expression vector pRSET-A, then transform to E.coli BL21(DE3)for expression under induction of IPTG. Purify the expressed product by Ni-NTA afinity column chromatography.
The target protein in a soluble form was successfully expressed in E.coli. Homogenous TCS mutant protein was obtained after purification of expressed product.
TCS mutant gene TCS(RL28-29CG); is succ-essfully constructed and expressed.
构建并表达天花粉蛋白（TCS）基因突变体，纯化表达产物。
通过计算机建模预测TCS分子上潜在的抗原决定簇并诱导定点突变。以栝楼基因组DNA为模板，通过PCR扩增基因突变体TCS（RL28 - 29CG），并插入表达载体pRSET - A，然后转化至大肠杆菌BL21（DE3）中，在IPTG诱导下进行表达。通过Ni - NTA亲和柱层析纯化表达产物。
目标蛋白以可溶形式在大肠杆菌中成功表达。表达产物纯化后获得了均一的TCS突变蛋白。
成功构建并表达了TCS突变基因TCS（RL28 - 29CG）。
