PRAS40与14-3-3蛋白各亚型相互作用的酵母双杂交系统检测

Study of Interaction Between PRAS40 and 14-3-3 Proteins by Using Yeast Two-hybrid System

PRAS40是近几年新发现的Akt作用底物,14-3-3结合蛋白。为确定PRAS40与14-3-3蛋白7种亚基间相互作用关系,利用gateway方法构建用于酵母双杂交系统的诱饵质粒pEG-PRAS40及转录激活质粒pJG-PRAS40,将PRAS40和14-3-3各亚型质粒分别作为诱饵蛋白质粒及转录激活质粒共转化酵母细胞EGY48,通过氨基酸营养缺陷生长实验及β-半乳糖苷酶显色反应分析两种蛋白相互作用程度。酶切鉴定证实成功地构建了pEG-PRAS40和pJG-PRAS40质粒,酵母双杂交实验结果显示PRAS40可以和14-3-3亚型tau,beta,zeta及epsilon相结合,epsilon较强,beta和zeta次之,tau较弱。此结果将为深入研究PRAS40与14-3-3蛋白生物学功能及发现药物靶标奠定基础。

PRAS40, a proline-rich Akt substrate of 40kD, is 14-3-3 binding protein. To study the interaction between PRAS40 and 14-3-3 isoforms, We constructed the expression vector pEG-PRAS40 （DNA-binding plasmid） and pJG-PRAS40 （transcriptional activity plasmid） in yeast using gateway cloning technology, then the plasmid of pEG- PRAS40/pJG-PRAS40 was co-transformed into yeast EGY48 strain with each pJG-14-3-3/pEG-14-3-3 isoform plasmid. The co-transformation were tested by nutrition limitation growth analysis, β-galactosidase color assay was used to study the interaction degree between PRAS40 and 14- 3-3 isoforms. We confirmed successfully the construction of pJG-PRAS40 and pEG-PRAS40 with enzyme digestion, four 14-3-3 isoforms were found interacting with PRAS40 using yeast two-hybrid assay, the interaction degree of Epsilon was stronger than beta and zeta, tau was the weakest. Our result will be used to further study the biological function of PRAS40 and 14-3-3 as new drug target.