Construction of eukaryotic expression vectors of carboxyl terminus and amino terminus of kinase suppressor of Ras (KSR) and their expression in 293T cell line / 中国肺癌杂志

ABSTRACT

<p><b>BACKGROUND</b>The present experimental data have showed that the function of kinase suppressor of Ras (KSR) is mainly as a scaffold protein that coordinates the assembly of a multiprotein complex containing MAPK and its upstream regulators. But whether KSR has kinase activity is still the point of argument until now. The aim of this study is to construct eukaryotic expression vectors of carboxyl terminus and amino terminus of KSR and to detect their expression in 293T cell line.</p><p><b>METHODS</b>N-KSR and C-KSR were amplified by PCR. The eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR were constructed by gene recombination technique and the recombinant plasmids were verified by restriction enzyme analysis and sequencing. Then positive clones were transfected into 293T cell line. Expression of target proteins was analyzed by Western blot.</p><p><b>RESULTS</b>The sequences and open read frames of the two vectors were both completely concordant with experiment design. The target proteins could be observed in transfected 293T cells by Western blot.</p><p><b>CONCLUSIONS</b>Eukaryotic expression vectors of pCMV-Tag2b-N-KSR and pCMV-Tag2b-C-KSR are successfully constructed, and they can be expressed in 293T cells. It provides an experimental base for further research work.</p>