| Efficient condensed-phase production of perdeuterated soluble and membrane proteins. | |
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MedLine Citation:
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PMID: 20333498 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Protein perdeuteration approaches have tremendous value in protein NMR studies, but are limited by the high cost of perdeuterated media. Here, we demonstrate that E. coli cultures expressing proteins using either the condensed single protein production method (cSPP), or conventional pET expression plasmids, can be condensed prior to protein expression, thereby providing high-quality (2)H, (13)C, (15)N-enriched protein samples at 2.5-10% the cost of traditional methods. As an example of the value of such inexpensively-produced perdeuterated proteins, we produced (2)H, (13)C, (15)N-enriched E. coli cold shock protein A (CspA) and EnvZb in 40x condensed phase media, and obtained NMR spectra suitable for 3D structure determination. The cSPP system was also used to produce (2)H, (13)C, (15)N-enriched E. coli plasma membrane protein YaiZ and outer membrane protein X (OmpX) in condensed phase. NMR spectra can be obtained for these membrane proteins produced in the cSPP system following simple detergent extraction, without extensive purification or reconstitution. This allows a membrane protein's structural and functional properties to be characterized prior to reconstitution, or as a probe of the effects of subsequent purification steps on the structural integrity of membrane proteins. We also provide a standardized protocol for production of perdeuterated proteins using the cSPP system. The 10-40 fold reduction in costs of fermentation media provided by using a condensed culture system opens the door to many new applications for perdeuterated proteins in spectroscopic and crystallographic studies. |
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Authors:
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William M Schneider; Yuefeng Tang; S Thangminlal Vaiphei; Lili Mao; Melissa Maglaqui; Masayori Inouye; Monica J Roth; Gaetano T Montelione |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural Date: 2010-03-24 |
Journal Detail:
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Title: Journal of structural and functional genomics Volume: 11 ISSN: 1570-0267 ISO Abbreviation: J. Struct. Funct. Genomics Publication Date: 2010 Jun |
Date Detail:
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Created Date: 2010-06-14 Completed Date: 2010-09-21 Revised Date: 2011-05-05 |
Medline Journal Info:
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Nlm Unique ID: 101128185 Medline TA: J Struct Funct Genomics Country: Netherlands |
Other Details:
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Languages: eng Pagination: 143-54 Citation Subset: IM |
Affiliation:
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Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Bacterial Outer Membrane Proteins
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chemistry,
genetics,
metabolism* Deuterium / chemistry* Escherichia coli / chemistry, genetics, metabolism* Escherichia coli Proteins / chemistry, genetics, metabolism* Gene Expression Genetic Vectors Heat-Shock Proteins / chemistry, genetics, metabolism* Membrane Proteins / chemistry, genetics, metabolism* Multienzyme Complexes / chemistry, genetics, metabolism* Nuclear Magnetic Resonance, Biomolecular Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization |
| Grant Support | |
ID/Acronym/Agency:
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R01 GM070837/GM/NIGMS NIH HHS; R01 GM070837-04/GM/NIGMS NIH HHS; T32 A1007403//PHS HHS; T32 GM08360/GM/NIGMS NIH HHS; U54 GM074958/GM/NIGMS NIH HHS; U54 GM75026/GM/NIGMS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Outer Membrane Proteins; 0/CspA protein, E coli; 0/Escherichia coli Proteins; 0/Heat-Shock Proteins; 0/Membrane Proteins; 0/Multienzyme Complexes; 7782-39-0/Deuterium; EC 2.7.3.-/envZ protein, E coli |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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