High Level Soluble Expression and ATPase Characterization of Human Heat Shock Protein GRP78.
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.
Chongqing Medical University, Key Laboratory of Molecular Biology of Infectious Disease, YiXueYuanlu-1, Chongqing, 400016, P. R. China. wangdq@cqmu.edu.cn.
Shuang
S
Wu
Hongpeng
H
Zhang
Miao
M
Luo
Ke
K
Chen
Wei
W
Yang
Lei
L
Bai
Ailong
A
Huang
Deqiang
D
Wang
D000251:Adenosine Triphosphatases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases