title: Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.
abstract: Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins.
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Xiao-Jing
authors.initials: XJ
authors.lastname: Li
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Jin-Ling
authors.initials: JL
authors.lastname: Liu
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Dong-Sheng
authors.initials: DS
authors.lastname: Gao
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Wen-Yan
authors.initials: WY
authors.lastname: Wan
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Yang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Yong-Tao
authors.initials: YT
authors.lastname: Li
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Chang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China.
authors.forename: Chuan-Qing
authors.initials: CQ
authors.lastname: Wang
authors.affiliation: College of Animal Science and Veterinary Medicine, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, Henan 450002, China. Electronic address: zhaoj@henau.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000363:Agaricales / Q000737:chemistry; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000191:economics; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli; D037102:Lectins / Q000737:chemistry; D008969:Molecular Sequence Data; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D012685:Sepharose / Q000737:chemistry
chemical_list: D037102:Lectins; D011993:Recombinant Fusion Proteins; D012685:Sepharose; D010450:Endopeptidases; C099488:TEV protease
keywords: Affinity purification; Lectin-tag; Mushroom Laetiporus sulphureus; Sepharose-binding; Tobacco etch virus protease