人泛素结合酶9的原核表达与纯化

Expression and Purification of Human Ubiquitin Conjugating Enzyme 9

目的:表达和纯化高纯度的人泛素结合酶9(hUBC9)。方法:将hUBC9基因克隆到原核表达载体pGEX-6p-1上并转化大肠杆菌BL21(DE3),于30℃、1mmol/LIPTG诱导4h,表达GST-hUBC9融合蛋白。用Glutathione-Sepharose4B柱分离GST-hUBC9融合蛋白,用鼻病毒3C蛋白水解酶切去GST-hUBC9融合蛋白的GST标签,再用FPLC分子筛层析法进一步纯化hUBC9。结果和结论:获得了重组表达质粒GST-hUBC9并在大肠杆菌中可溶性表达,经亲和层析、酶切、分子筛层析后获得了可溶的、高纯度的hUBC9,为进一步研究hUBC9的功能和结构奠定了基础。

Objective： To express and spurify human ubiquitin conjugating enzyme9（hUBC9）. Methods： The gene of hUBC9 was cloned into expression vector pGEX-6p-1, expressed fusion GST-hUBC9 protein in E.coli BI21（DE3） at 30℃ with 1 mmol/L IPTG for 4 hours. The supernatants lysed by sonication and clarified by centrifugation were passed over Glutathione-Sepharose 4B column and load on the SuperdexTM G200 column, and then the soluble hUBC9 protein was putiffed. Results ＆ Conclusion： The fusion GST-hUBC9 protein was successfully expressed in E.coli BI21（DE3） and purified by Glutathione-Sepharose 4B column. After hydrolyzed by 3C and purified by SuperdexTM G200 column, high quality hUBC9 was obtained.