title: A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease.
abstract: Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16-TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16-TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-γ (His-BoIFN-γ) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 1.3 × 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-γ (rBoIFN-γ) was purified to 98.3% purity with 63% recovery. The rBoIFN-γ had an antiviral activity of 1.6 × 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16-TEVp may become a universal tool for recombinant protein purification.
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Guang-Ya
authors.initials: GY
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Zhen-Zhen
authors.initials: ZZ
authors.lastname: Xiao
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Hui-Peng
authors.initials: HP
authors.lastname: Lu
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Yang-Yang
authors.initials: YY
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Xiao-Hui
authors.initials: XH
authors.lastname: Zhou
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Tan
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Xin-Yu
authors.initials: XY
authors.lastname: Zhang
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Xia
authors.affiliation: College of Veterinary Medicine, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou, 225009, China. Electronic address: sunhyzu@163.com.
authors.forename: Huai-Chang
authors.initials: HC
authors.lastname: Sun
mesh_terms: D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000737:chemistry*; D010942:Plant Viruses / Q000201:enzymology / Q000235:genetics*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011993:Recombinant Fusion Proteins; D007371:Interferon-gamma; D010450:Endopeptidases; C099488:TEV protease
keywords: Active protein aggregate; Protein purification; Self-assembling peptide; Tobacco etch virus protease