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

Shuang Wu1,2#, Hongpeng Zhang1,2#, Miao Luo3#, Ke Chen1, Wei Yang2, Lei Bai1, Ailong Huang1, and Deqiang Wang1,2*

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

DOI: 10.1134/S0006297917020109