Source:http://linkedlifedata.com/resource/pubmed/id/16594652
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
14
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| pubmed:dateCreated |
2006-4-5
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| pubmed:abstractText |
A simple, rapid, in vitro cell-free protein expression system, Expressway NMR, is introduced and used to express the small ubiquitin-related modifier protein SUMO-1. This 12 kDa molecule is challenging for NMR as it has limited solubility and requires relatively high salt (200 mM) for stability in solution. Starting with the gene, the cell-free system, and milligram amounts of nitrogen-15 isotopically enriched amino acids, sufficient protein is produced in 4 h to obtain a high-resolution 2D HSQC spectrum of the protein in 40 min. This time would be closer to 10 min with the aid of a higher sensitivity salt-tolerant cryogenic NMR probe. With all protein purification steps included, and aggressive data processing using the filter diagonalization method (FDM), it is but 6 h from gene to heteronuclear single quantum coherence (HSQC). As the cell-free system is nearly background-free, it is also possible to work with the crude reaction mixture, in which case only a total of 5 h is required. Sample stability over time, whether crude extract or purified, was notable, with no significant change in the 15N-1H HSQC spectrum over 6 months at 4 degrees C (300 muM, pH 6.1, capped NMR tube). The combination of a turnkey, high-yield, protease-free in vitro protein expression system, an optimized sensitivity-enhanced HSQC pulse sequence, and FDM processing makes this scheme an attractive first step to rapidly assess the suitability of proteins for complete solution structure determination.
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| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:status |
PubMed-not-MEDLINE
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| pubmed:month |
Apr
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| pubmed:issn |
0002-7863
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
12
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| pubmed:volume |
128
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
4508-9
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| pubmed:dateRevised |
2008-1-17
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| pubmed:year |
2006
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| pubmed:articleTitle |
From gene to HSQC in under five hours: high-throughput NMR proteomics.
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| pubmed:affiliation |
Invitrogen Corporation, 1610 Faraday Avenue, Carlsbad, California 92008, USA. ajshaka@uci.edu
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| pubmed:publicationType |
Journal Article
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