Background: 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.
Conclusions: Two mutant KSR genes are successfully constructed. It provides experimental basement for further functional research of KSR.