天花粉蛋白Y14F/R22L定点突变及其活性研究  

Y14F/R22L SiteDirected Mutagenesis of Trichosanthinand Study on Its Activity

作　　者：姚宏兵[1,2] 吴伸[1,2] 董贻诚[1,2] 邵鹏柱[1,2] 

机构地区：[1]中国科学院生物物理研究所 [2]香港中文大学生化系

出　　处：《中国生物化学与分子生物学报》1998年第3期264-268,共5页Chinese Journal of Biochemistry and Molecular Biology

基　　金：香港研究基金委员会CUHK11/91基金

摘　　要：利用多聚酶链式反应（ＰＣＲ）技术，对天然天花粉蛋白（ｎＴＣＳ）基因在Ｔｙｒ１４和Ａｒｇ２２两个保守残基处同时进行定点突变，即Ｔｙｒ１４变成Ｐｈｅ，Ａｒｇ２２变成Ｌｅｕ，然后克隆到ｐＥＴ－８ｃ高效表达载体上，构建成重组质粒ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ．经序列分析，定点突变的结果与预先设计的完全一致，突变后的天花粉蛋白命名为Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ．将ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ转化到Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３，ｐＬｙｓＳ）中，进行表达．经ＣＭ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱纯化，ＳＤＳ－ＰＡＧＥ鉴定，纯度可达９０％．ＲＩＰ活性测定显示，Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ的活性比ｎＴＣＳ降低了７．５倍，活性变化不显著，因此，ＴＣＳ的Ｔｒｙ１４和Ａｒｇ２２对维持其活性部位构象并不是必需的．但由于Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ在Ｅ．ｃｏｌｉ中的表达量与ｎＴＣＳ相比明显下降，因此，Ｔｙｒ１４和Ａｒｇ２２可能与ＴＣＳ翻译后的折叠有关．Tyr14 and Arg22 of nTCS were substituted respectively by Phe and Leu using PCR for sitedirected mutagenesis.The mutated gene was cloned into pET8c to form recombinant plasmid pETY14F/R22L.The DNA sequence analysis showed that the result of sitedirected mutagenesis was in accordance with original design,and the mutant was named Y14F/R22L TCS.The recombinant plasmid was transformed into BL21(DE3,pLysS) and then expressed.After purification by CMSepharose CL6B column,the mutated TCS was found to be homogeneous.Compared with nTCS,its RIP activity varied not so distinctly and was only reduced by 75 fold.Therefore Tyr14 and Arg22 of TCS are not essential to maintain its active site conformation.However,yield of Y14F/R22L TCS in E.coli was significantly low.This indicates that Tyr14 and Arg22 may play a role in the correct folding of trichosanthin.

关 键 词：天花粉蛋白 定点突变 PCR 序列分析 

分 类 号：Q946.1[生物学—植物学] Q343.13