Cell-free methods to produce structurally intact mammalian membrane proteins
| Autor: | Noboru Ohsawa, Yohei Ishibashi, Mikako Shirouzu, Yoshiaki Kawano, Tomomi Kimura-Someya, Shigeyuki Yokoyama, Naoko Shinya, Masaki Yamamoto, Takaho Terada, Kunio Hirata, Yoshio Hirabayashi, Yoshiko Ishizuka-Katsura, Takehiro Shinoda, Kaori Ito, Taisuke Tomita |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Biology Crystallography X-Ray Article Cell-free system 03 medical and health sciences Enterotoxins Column chromatography Protein biosynthesis Animals Chemical Precipitation Humans Claudin-4 Integral membrane protein Mammals Multidisciplinary Cell-Free System Peripheral membrane protein Cell Membrane Membrane Proteins Lipids Protein Subunits 030104 developmental biology Membrane Biochemistry Membrane protein Solubility Glucosyltransferases Ultracentrifuge Amyloid Precursor Protein Secretases Ultracentrifugation Subcellular Fractions |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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