title: Autoprocessing: an essential step for expression and purification of enterovirus 71 3C(pro) in Escherichia coli.
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 (3C(pro)) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C(pro) 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 3C(pro). The EV71 3C(pro) had a Km value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C(pro) 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 3C(pro).
authors.affiliation: 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, ahuanqsq@imm.ac.cn.
authors.forename: Shuqiong
authors.initials: S
authors.lastname: Huang
authors.forename: Yanning
authors.initials: Y
authors.lastname: Lyu
authors.forename: Xianyun
authors.initials: X
authors.lastname: Qing
authors.forename: Weiwei
authors.initials: W
authors.lastname: Wang
authors.forename: Liang
authors.initials: L
authors.lastname: Tang
authors.forename: Kedi
authors.initials: K
authors.lastname: Cheng
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
mesh_terms: D000086042:3C Viral Proteases; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D029821:Enterovirus A, Human / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D059748:Proteolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases