High level soluble expression and ATPase characterization of human heat shock protein GRP78
- Authors: Wu S.1,2, Zhang H.1,2, Luo M.2, Chen K.1, Yang W.2, Bai L.1, Huang A.1, Wang D.1,2
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Affiliations:
- Key Laboratory of Molecular Biology of Infectious Disease
- Department of Laboratory Medicine
- Issue: Vol 82, No 2 (2017)
- Pages: 186-191
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151257
- DOI: https://doi.org/10.1134/S0006297917020109
- ID: 151257
Cite item
Abstract
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.
Keywords
About the authors
Shuang Wu
Key Laboratory of Molecular Biology of Infectious Disease; Department of Laboratory Medicine
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016; YiXueYuanlu-1, Chongqing, 400016
Hongpeng Zhang
Key Laboratory of Molecular Biology of Infectious Disease; Department of Laboratory Medicine
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016; YiXueYuanlu-1, Chongqing, 400016
Miao Luo
Department of Laboratory Medicine
Email: wangdq@cqmu.edu.cn
China, No. 61 JIANSHE Road, YuBei District, Chongqing, 401120
Ke Chen
Key Laboratory of Molecular Biology of Infectious Disease
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016
Wei Yang
Department of Laboratory Medicine
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016
Lei Bai
Key Laboratory of Molecular Biology of Infectious Disease
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016
Ailong Huang
Key Laboratory of Molecular Biology of Infectious Disease
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016
Deqiang Wang
Key Laboratory of Molecular Biology of Infectious Disease; Department of Laboratory Medicine
Author for correspondence.
Email: wangdq@cqmu.edu.cn
China, YiXueYuanlu-1, Chongqing, 400016; YiXueYuanlu-1, Chongqing, 400016