title: A novel self-cleavage system for production of soluble recombinant protein in Escherichia coli.
abstract: Many approaches for generating large quantities of recombinant protein in Escherichia coli fuse the protein of interest to a protein tag to enhance solubility and improve recovery. However, the fusion tags can confound downstream applications, as the fusion partner can alter the structure and biological activity of the recombinant protein and proteolytic removal of the fusion tags can be expensive. Here we describe a new system for production of native proteins in E. coli that allows for removal of the fusion tag via intracellular self-cleavage by the human rhinovirus 3C (HRV3C) protease. This system allows for parallel cloning of target protein coding sequences into six different expression vectors, each with a different fusion partner tag to enhance solubility during induction. Temperature-regulated expression of the HRV3C protease allows for intracellular removal of the fusion tag following induction, and the liberated recombinant protein can be purified by affinity chromatography by virtue of a short six-histidine tag. This system will be an attractive approach for the expression and purification of recombinant proteins free of solubility-enhancing fusion tags, and should be amenable to high-throughput applications.
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang Province, PR China; The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
authors.forename: Yufei
authors.initials: Y
authors.lastname: Feng
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
authors.forename: Qingyuan
authors.initials: Q
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
authors.forename: Tao
authors.initials: T
authors.lastname: Yang
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
authors.forename: Encheng
authors.initials: E
authors.lastname: Sun
authors.affiliation: The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China.
authors.forename: Junping
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang Province, PR China.
authors.forename: Dongfang
authors.initials: D
authors.lastname: Shi
authors.affiliation: Department of Basic Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, 59 Mucai Street, Xiangfang District, Harbin 150030, Heilongjiang Province, PR China; The Key Laboratory of Veterinary Public Health, Ministry of Agriculture, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Maduan Street, Nangang District, Harbin 150001, Heilongjiang Province, PR China. Electronic address: dlwu@hvri.ac.cn.
authors.forename: Donglai
authors.initials: D
authors.lastname: Wu
mesh_terms: D000086042:3C Viral Proteases; D003001:Cloning, Molecular / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases
keywords: Escherichia coli (E. coli); Human rhinovirus 3C protease (HRV3C); Protein expression; Self-cleavage