title: A Novel and Fast Purification Method for Nucleoside Transporters.
abstract: Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.
authors.affiliation: Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, College of Environmental Science and Engineering, Nankai UniversityTianjin, China; Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of LeedsLeeds, UK.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds Leeds, UK.
authors.forename: Maren
authors.initials: M
authors.lastname: Thomsen
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of LeedsLeeds, UK; Biomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett UniversityLeeds, UK.
authors.forename: Vincent L G
authors.initials: VL
authors.lastname: Postis
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds Leeds, UK.
authors.forename: Amelia
authors.initials: A
authors.lastname: Lesiuk
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds Leeds, UK.
authors.forename: David
authors.initials: D
authors.lastname: Sharples
authors.affiliation: Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, College of Environmental Science and Engineering, Nankai University Tianjin, China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of LeedsLeeds, UK; Department of Molecular Biology and Biochemistry, College of Life Sciences, Nankai UniversityTianjin, China; State Key Laboratory of Medicinal Chemical Biology, Nankai UniversityTianjin, China.
authors.forename: Mark
authors.initials: M
authors.lastname: Bartlam
authors.affiliation: Faculty of Biological Sciences, Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of LeedsLeeds, UK; Department of Molecular Biology and Biochemistry, College of Life Sciences, Nankai UniversityTianjin, China; Division of Biochemistry, Department of Biosciences, University of HelsinkiHelsinki, Finland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
keywords: expression; membrane protein; nucleoside transporters; purification; vector construction