Wu Shuang, Zhang Hongpeng, Luo Miao, Chen Ke, Yang Wei, Bai Lei, Huang Ailong, Wang Deqiang
Chongqing Medical University, Key Laboratory of Molecular Biology of Infectious Disease, YiXueYuanlu-1, Chongqing, 400016, P. R. China.
Biochemistry (Mosc). 2017 Feb;82(2):186-191. doi: 10.1134/S0006297917020109.
Human GRP78 has been shown to promote cancer progression and is regarded as a novel target for anticancer drugs. However, generation of recombinant full-length GRP78 remains challenging. This report demonstrates that E. coli autoinduction is an excellent method for the preparation of active recombinant GRP78 protein. The final yield was approximately 50 mg/liter of autoinduction culture. Gel-filtration experiments confirmed that the chaperone is a monomer. The purified human GRP78 catalyzed the conversion of ATP to ADP without requiring metal ions as cofactors. Three mutants, T38A, T229A, and S300A, exhibited much lower activity than wild-type GRP78, indicating that the active sites of the ATPase are located at the negatively charged cavity. Three mutants in the negatively charged cavity region dramatically reduced GRP78 activity, further confirming the region as the site of ATPase activity.
已证明人类GRP78可促进癌症进展，被视为抗癌药物的新靶点。然而，重组全长GRP78的生成仍然具有挑战性。本报告表明，大肠杆菌自诱导是制备活性重组GRP78蛋白的一种出色方法。最终产量约为每升自诱导培养物50毫克。凝胶过滤实验证实该伴侣蛋白为单体。纯化的人类GRP78催化ATP转化为ADP，无需金属离子作为辅助因子。三个突变体T38A、T229A和S300A的活性远低于野生型GRP78，表明ATP酶的活性位点位于带负电荷的腔中。带负电荷腔区域的三个突变体显著降低了GRP78活性，进一步证实该区域为ATP酶活性位点。
