| High-throughput fluorescent-based optimization of eukaryotic membrane protein overexpression and purification in Saccharomyces cerevisiae. | |
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MedLine Citation:
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PMID: 17709746 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Eukaryotic membrane proteins are often difficult to produce in large quantities, which is a significant obstacle for further structural and biochemical investigation. Based on the analysis of 43 eukaryotic membrane proteins, we present a cost-effective high-throughput approach for rapidly screening membrane proteins that can be overproduced to levels of >1 mg per liter in Saccharomyces cerevisiae. We find that 70% of the well expressed membrane proteins tested in this system are stable, targeted to the correct organelle, and monodisperse in either Fos-choline 12 (FC-12) or n-dodecyl-beta-D-maltoside. We illustrate the advantage of such an approach, with the purification of monodisperse human and yeast nucleotide-sugar transporters to unprecedented levels. We estimate that our approach should be able to provide milligram quantities for at least one-quarter of all membrane proteins from both yeast and higher eukaryotic organisms. |
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Authors:
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Simon Newstead; Hyun Kim; Gunnar von Heijne; So Iwata; David Drew |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2007-08-20 |
Journal Detail:
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Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 104 ISSN: 0027-8424 ISO Abbreviation: Proc. Natl. Acad. Sci. U.S.A. Publication Date: 2007 Aug |
Date Detail:
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Created Date: 2007-08-29 Completed Date: 2007-10-17 Revised Date: 2013-06-06 |
Medline Journal Info:
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Nlm Unique ID: 7505876 Medline TA: Proc Natl Acad Sci U S A Country: United States |
Other Details:
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Languages: eng Pagination: 13936-41 Citation Subset: IM |
Affiliation:
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Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, United Kingdom. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Gene Expression Regulation, Fungal Genes, Reporter Membrane Proteins / genetics, isolation & purification, metabolism* Microscopy, Confocal Plasmids Recombinant Fusion Proteins / metabolism Recombinant Proteins / genetics, isolation & purification, metabolism Saccharomyces cerevisiae / metabolism* Saccharomyces cerevisiae Proteins / genetics, isolation & purification, metabolism |
| Chemical | |
Reg. No./Substance:
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0/Membrane Proteins; 0/Recombinant Fusion Proteins; 0/Recombinant Proteins; 0/Saccharomyces cerevisiae Proteins |
| Comments/Corrections | |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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