title: Cell-free methods to produce structurally intact mammalian membrane proteins.
abstract: The crystal structures of four membrane proteins, from bacteria or a unicellular alga, have been solved with samples produced by cell-free protein synthesis. In this study, for mammalian membrane protein production, we established the precipitating and soluble membrane fragment methods: membrane proteins are synthesized with the Escherichia coli cell-free system in the presence of large and small membrane fragments, respectively, and are simultaneously integrated into the lipid environments. We applied the precipitating membrane fragment method to produce various mammalian membrane proteins, including human claudins, glucosylceramide synthase, and the γ-secretase subunits. These proteins were produced at levels of about 0.1-1.0 mg per ml cell-free reaction under the initial conditions, and were obtained as precipitates by ultracentrifugation. Larger amounts of membrane proteins were produced by the soluble membrane fragment method, collected in the ultracentrifugation supernatants, and purified directly by column chromatography. For several proteins, the conditions of the membrane fragment methods were further optimized, such as by the addition of specific lipids/detergents. The functional and structural integrities of the purified proteins were confirmed by analyses of their ligand binding activities, size-exclusion chromatography profiles, and/or thermal stabilities. We successfully obtained high-quality crystals of the complex of human claudin-4 with an enterotoxin.
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Takehiro
authors.initials: T
authors.lastname: Shinoda
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Shinya
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Ito
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Ishizuka-Katsura
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Ohsawa
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
authors.forename: Takaho
authors.initials: T
authors.lastname: Terada
authors.affiliation: RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Hirata
authors.affiliation: RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Kawano
authors.affiliation: RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Tomita
authors.affiliation: Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ishibashi
authors.affiliation: Laboratory for Molecular Membrane Neuroscience, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Hirabayashi
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Tomomi
authors.initials: T
authors.lastname: Kimura-Someya
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.affiliation: RIKEN Systems and Structural Biology Center, Yokohama 230-0045, Japan.|RIKEN Structural Biology Laboratory, Yokohama 230-0045, Japan.
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D011232:Chemical Precipitation; D062506:Claudin-4 / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004768:Enterotoxins / Q000737:chemistry / Q000378:metabolism; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008322:Mammals / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014461:Ultracentrifugation / Q000379:methods*
chemical_list: D062506:Claudin-4; D004768:Enterotoxins; D008055:Lipids; D008565:Membrane Proteins; D021122:Protein Subunits; C035598:enterotoxin, Clostridium; D005964:Glucosyltransferases; C023246:ceramide glucosyltransferase; D053829:Amyloid Precursor Protein Secretases