Ras激酶抑制子KSR定点突变、表达和鉴定

背景与目的 Ras to MAPK通路在生长、发育信号传递中起关键的作用。Ras激酶抑制基因（KSR）是该通路中的一个重要组分，其功能主要是作为一个支架蛋白协调装配包含有丝分裂原活化蛋白激酶（MAPK）及其上游调控子的多蛋白复合体。已有的研究表明KSR有许多磷酸化位点，这些位点磷酸化状态的改变与外界信号刺激有着极为密切的关系。利用定点突变技术可以准确地改变基因特定碱基序列，获得突变蛋白质。本研究的目的是应用定点突变技术构建突变型KSR，并将其瞬时转染293T细胞所表达蛋白纯化和鉴定，为进一步研究KSR生化作用机制提供理论和实验依据。方法 以本实验室自己构建的pCMV-Tag2b-KSR质粒为模板，应用本实验室改进的QuikChang^tm定点突变方法，引入KSR12个突变位点，构建突变型pCMV-Tag2b-KSR，并将其瞬时转染293T细胞进行蛋白表达，亲和胶纯化并经SDS-PAGE和蛋白质印迹鉴定。结果 成功构建了2个突变型KSR基因，经DNA序列分析突变碱基，证实位点正确，并经过瞬时转染293T细胞表达出突变体蛋白，纯化蛋白经SDS-PAGE和蛋白质印迹鉴定与实验设计完全一致。结论 成功构建了2个具有不同突变位点的突变型KSR，为以后进行蛋白功能研究提供实验基础。

Construction, expression and purification of kinase suppressor of Ras, KSR

Background and objective Ras to MAPK pathway plays a critical role in the transmission of many growth and developmental signals. A new component of this pathway which is termed kinase suppressor of Ras (KSR) was found in 1995. KSR is as a scaffolding protein that coordinates the assembly of a multiprotein complex containing mitogen-activated protein kinase (MAPK) and its upstream regulators. It has been proven that KSR has many phosphorylation sites, and phosphorylation state changes response to signaling events. Site-directed mutagenesis can precisely change the base sequence and get mutant proteins. The aim of this study is to construct two mutant proteins of KSR by using site-directed mutagenesis, and to express and purify them, therefore to provide basement for studying the functional and biochemical mechanisms of KSR. Methods Site-directed mutagenesis of pCMV-Tag2b-KSR gene was performed by modified QuikChangtm site-directed mutagenesis kit method. Two pairs of mutagenic primers were synthesized in vitro and two mutations desired, the recombinant plasmids were verified by restriction enzyme analysis and DNA sequencing. Then positive clones were transfected into 293T cell line. The purified mutant proteins were analyzed by Western blot. Results Two mutants were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the mutant genes were completely concordant with experiment design, which could be used to be transfected into 293T cell line. The purified mutants were identified by Western blot. Conclusion Two mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.