High Level Soluble Expression and ATPase Characterization of Human Heat Shock Protein GRP78

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Shuang Wu^1,2#, Hongpeng Zhang^1,2#, Miao Luo^3#, Ke Chen¹, Wei Yang², Lei Bai¹, Ailong Huang¹, and Deqiang Wang^1,2*

¹Chongqing Medical University, Key Laboratory of Molecular Biology of Infectious Disease, YiXueYuanlu-1, 400016 Chongqing, People’s Republic of China; E-mail: wangdq@cqmu.edu.cn

²Chongqing Medical University, Department of Laboratory Medicine, YiXueYuanlu-1, 400016 Chongqing, People’s Republic of China

³People’s Hospital of YuBei District, Department of Laboratory Medicine, No. 61 JIANSHE Road, YuBei District, 401120 Chongqing, People’s Republic of China

^# These authors contributed equally to this work.

^* To whom correspondence should be addressed.

Received May 17, 2016; Revision received September 29, 2016

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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.

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KEY WORDS: GRP78, ATPase, autoinduction, monomer, active site

DOI: 10.1134/S0006297917020109

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