ArticleScientific reports2016
Cell-free methods to produce structurally intact mammalian membrane proteins.
Article in Scientific reports, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 40 papers.
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Who cites it
40 citing papers in PubMed, 75 citations in OpenAlex.
- Advances in Crystallography and Cryo-Electron Microscopy for the Study of Membrane Proteins.Advances in experimental medicine and biology · 2026Review
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- Applications of the wheat germ cell-free protein synthesis system in plant biochemical studies.Plant biotechnology (Tokyo, Japan) · 2024Article
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- Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins.Communications biology · 2024Article
- Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion.Communications biology · 2023Article
- Opportunities to accelerate extracellular vesicle research with cell-free synthetic biology.Journal of extracellular biology · 2023Article
- Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives.Bioengineering & translational medicine · 2023Review
- Crystal Structure of an Archaeal Tyrosyl-tRNA Synthetase Bound to Photocaged L-Tyrosine and Its Potential Application to Time-Resolved X-ray Crystallography.International journal of molecular sciences · 2022Article
- Review
- High-throughput cell-free screening of eukaryotic membrane protein expression in lipidic mimetics.Protein science : a publication of the Protein Society · 2022Article
- Human Claudin-7Frontiers in molecular biosciences · 2022Article
- Cell-Free Membrane Protein Expression into Hybrid Lipid/Polymer Vesicles.Methods in molecular biology (Clifton, N.J.) · 2022Article
- News Feature: Biology research, no cells required: By borrowing the machinery of life from broken cells, researchers are producing novel proteins, cheap lab reagents, and just-add-water vaccines.Proceedings of the National Academy of Sciences of the United States of America · 2021Article
- Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems.Journal of visualized experiments : JoVE · 2021Article
- Engineering spatiotemporal organization and dynamics in synthetic cells.Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology · 2021Review
- Structural basis forProceedings of the National Academy of Sciences of the United States of America · 2021Article
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Authors and funding
15 authors at 5 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
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.
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