人RAF1-RAS结合域蛋白的纯化及功能验证

Purification of human RAF1-RAS binding domain protein and function verification

目的纯化人RAF1(RAF proto-oncogene serine/threonine-protein kinase)-RAS(rat sarcoma)结合域(RAF1-RAS binding domain,RAF1-RBD)蛋白并验证其功能。方法采用PCR法以人乳腺文库cDNA为模板扩增获得RAF1-RBD序列,将其插入至大肠杆菌表达载体pGEX-KG(带GST标签),获得pGEX-KG-RAF1-RBD重组质粒;转化该质粒至感受态细菌BL21(DE3),小量诱导并通过Western印迹法检测其表达;然后采用GST微珠纯化该蛋白;利用GST pull-down技术验证其与HRAS(Harvey rat sarcoma viral oncogene homolog)蛋白相互作用。结果双酶切鉴定及基因测序结果均表明成功构建重组质粒pGEX-KG-RAF1-RBD;Western印迹分析及SDS-PAGE方法检测结果说明重组蛋白相对分子质量(Mr)约42 000,与预期大小一致;GST pull-down技术证明RAF1-RBD与HRAS在体外能够结合。结论纯化的人RAF1-RBD蛋白与HRAS在体外存在相互作用。

Objective To purify human RAF1(RAF proto-oncogene serine/threonine-protein kinase)-RBD protein and verify its function. Methods RAF1-RBD sequence was amplified by PCR using human mammary gland library cDNA as the template and inserted into E.coli expression vector pGEX-KG(with GST tag)to obtain recombinant plasmid of pGEX-KG-RAF1-RBD. The recombinant plasmid was transformed into competent bacteria BL21(DE3),and its expression was detected by small amount induction and Western blotting. The protein was purified by GST beads and its interaction with Harvey rat sarcoma viral oncogene homolog(HRAS)protein was tested by GST pull-down technique.Results The recombinant plasmid pGEX-KG-RAF1-RBD was successfully constructed and verified by double digestion and gene sequencing. Western blotting analysis and SDS-PAGE analysis showed that the relative molecular mass(Mr)of the recombinant protein was about 42 000,which was of the expected size. GST pull-down technique proved that RAF1-RBD could bind to HRAS in vitro. Conclusion The purified human RAF1-RBD protein interacts with HRAS in vitro.