title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters.
abstract: Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Cheng
authors.initials: C
authors.lastname: Ma
authors.affiliation: Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin, China.|Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Hao
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Gerard
authors.initials: G
authors.lastname: Huysmans
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Amelia
authors.initials: A
authors.lastname: Lesiuk
authors.affiliation: Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, United Kingdom.
authors.forename: Per
authors.initials: P
authors.lastname: Bullough
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: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.|College of Life Sciences, Nankai University, Tianjin, China.|State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin, China.
authors.forename: Mark
authors.initials: M
authors.lastname: Bartlam
authors.affiliation: Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot, Oxfordshire, United Kingdom.
authors.forename: Simon E
authors.initials: SE
authors.lastname: Phillips
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Canada.
authors.forename: James D
authors.initials: JD
authors.lastname: Young
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.|College of Life Sciences, Nankai University, Tianjin, China.|Division of Biochemistry, Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Baldwin
authors.affiliation: Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom.|Biomedicine Research Group, Faculty of Health and Social Sciences, Leeds Beckett University, Leeds, LS1 3HE, United Kingdom.
authors.forename: Vincent L G
authors.initials: VL
authors.lastname: Postis
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism*; D015870:Gene Expression; D023061:Gene Order; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D033703:Nucleoside Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D008670:Metals; D033703:Nucleoside Transport Proteins; D011993:Recombinant Fusion Proteins