title: Soluble expression and enzymatic activity evaluation of protease from reticuloendotheliosis virus.
abstract: The protease (PR) encoded by most retroviruses is deeply involved in the lifecycle and infection process of retroviruses by possessing the specificity necessary to correctly cleave the viral polyproteins and host cell proteins. However, as an important representative of avian retroviruses, the enzymatic properties of PR from reticuloendotheliosis virus (REV) have not been clearly documented. The recombinant PR, its mutant fused with a His-tag, and its substrate p18-p30 fused with a GST-tag were expressed in the Escherichia coli system as soluble enzymes. The soluble PR and p18-p30 were purified using Ni-NTA His Bind Resin and Glutathione Sepharose 4B, respectively. The enzymatic activity of PR was analyzed using the substrate of p18-p30. The expressed prokaryotic protease has enzyme activity that is dependent on such conditions as temperature, pH, and ions, and its activity can be inhibited by caspase inhibitor and the divalent metal ions Ca(2+) and Ni(2+). In addition, the key role of the residue Thr (amino acids 28) for the enzymatic activity of PR was identified. Furthermore, the caspase inhibitor Z-VAD-FMK was confirmed to inhibit the PR enzymatic activity of REV. For the first time, the PR of REV was expressed in the soluble form, and the optimal enzymatic reaction system in vitro was developed and preliminarily used. This study provides essential tools and information for further understanding the infection mechanism of REV and for the development of antiviral drugs treating retroviruses.
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: hufengggg@126.com.
authors.forename: Feng
authors.initials: F
authors.lastname: Hu
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: kriyazhao@hvri.ac.cn.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: qxl@hvri.ac.cn.
authors.forename: Xiaole
authors.initials: X
authors.lastname: Qi
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: cuihongyu@caas.cn.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Cui
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: ylg@hvri.ac.cn.
authors.forename: Yulong
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: ghl@hvri.ac.cn.
authors.forename: Honglei
authors.initials: H
authors.lastname: Gao
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: liucj93711@hvri.ac.cn.
authors.forename: Changjun
authors.initials: C
authors.lastname: Liu
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: yqw_19@yahoo.cn.
authors.forename: Yongqiang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: zhangyanping03@caas.cn.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China. Electronic address: likaihvri@163.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China. Electronic address: xmw@hvri.ac.cn.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, The Chinese Academy of Agricultural Sciences, Harbin 150001, China; National Engineering Research Center of Veterinary Biologics, Harbin, China. Electronic address: yfwang@hvri.ac.cn.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001356:Reticuloendotheliosis virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D010447:Peptide Hydrolases
keywords: Enzyme activity; Expression; Protease; Purification; Reticuloendotheliosis virus