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Autoprocessing: an essential step for expression and purification of enterovirus 71 3Cpro in Escherichia coli |
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| Authors: | Shuqiong Huang Yanning Lyu Xianyun Qing Weiwei Wang Liang Tang Kedi Cheng Wei Wang |
| Institution: | 1. Key Laboratory of Biosynthesis of Natural Products, State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Ministry of Health of PRC, Beijing, 100050, China 2. Institute for Infectious Disease and Endemic Disease Prevention and Control, Beijing Center for Disease Prevention and Control, Beijing, 100013, China 3. College of Life Science, Qufu Normal University, Qufu, 273165, China |
| Abstract: | 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. |
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