Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli
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A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3Cpro) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3Cpro partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3Cpro. The EV71 3Cpro had a Km value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3Cpro was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3Cpro.
KeywordsAutoprocessing Enterovirus 71 3C protease pET Trx fusion system Solid-phase activity assay Virus expression Virus purification
This study was financially supported by the National Science Foundation of China (Grant Nos. 30772677, 81072562); the Fundamental Research Funds for the Central Universities (Grant No. 2012N06); the National Science and Technology Project of China (Grant No. 2012ZX09301002).
- Zhang Y, Zhu Z, Yang W, Ren J, Tan X, Wang Y, Mao N, Xu S, Zhu S, Cui A, Zhang Y, Yan D, Li Q, Dong X, Zhang J, Zhao Y, Wan J, Feng Z, Sun J, Wang S, Li D, Xu W (2010) An emerging recombinant human enterovirus 71 responsible for the 2008 outbreak of hand foot and mouth disease in Fuyang city of China. Virol J 7:94PubMedCrossRefGoogle Scholar