Linked Life Data

20333498

Source:http://linkedlifedata.com/resource/pubmed/id/20333498

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2010-6-14
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
1570-0267
pubmed:author
pubmed:issnType
Electronic
pubmed:volume
11
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
143-54
pubmed:dateRevised
2011-5-5
pubmed:meshHeading
pubmed:year
2010
pubmed:articleTitle
Efficient condensed-phase production of perdeuterated soluble and membrane proteins.
pubmed:affiliation
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural