von Hippel-Lindau(VHL)的表达、纯化及结合活性分析

Expression,purification and binding activity analysis of von Hippel-Lindau( VHL)

目的纯化人von Hippel-Lindau(VHL)基因重组蛋白并进行功能鉴定。方法采用PCR从人乳腺c DNA中获得VHL基因序列,该序列被插入到原核表达载体p GEX-KG中,得到谷胱甘肽巯基转移酶(GST)-VHL重组质粒,将其转化至BL21(DE3)感受态细菌,经小量诱导后,采用SDS-PAGE和Western blot法检测该蛋白表达情况,使用GST微珠纯化该重组蛋白,利用GST pull-down技术验证其功能。结果获得的重组质粒可成功双酶切,基因测序表明VHL序列正确且无突变;将其转化至BL21(DE3)感受态细菌并小量诱导,纯化得到相对分子质量(Mr)约56 000的重组蛋白; GST pull-down技术证明GST-VHL重组蛋白在体外具有结合缺氧诱导因子1α(HIF-1α)的功能。结论纯化出GST-VHL重组蛋白并可与HIF-1α蛋白在体外结合。

Objective To purify recombinant protein of human von Hippel-Lindau（ VHL） and identify its function.Methods VHL gene sequence was amplified from human mammary c DNA using PCR and inserted into the prokaryotic expression vector p GEX-KG. Glutathione S-transferase-VHL（ GST-VHL） recombinant plasmid we obtained was converted into BL21（ DE3） sensitive bacteria to induce a small amount of GST-VHL protein. The expressed product was detected by SDS-PAGE and Western blot analysis. The recombinant protein was purified by GST beads and its function was verified by GST pull-down assay. Results The obtained recombinant plasmid could be successfully digested by double enzymes. Gene sequencing showed that the VHL sequence was correct and there was no mutation. The recombinant protein with approximately relative molecular mass（ Mr） 56 000 was purified by converting recombinant plasmid to BL21（ DE3） sensitive bacteria and inducing it in small quantities. GST pull-down assay verified that GST-VHL recombinant protein had the function of binding hypoxia inducible factor-1 α（ HIF-1 α） in vitro. Conclusion GST-VHL recombinant protein is purified and can combine with HIF-1α protein in vitro.