Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR View Full Text


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Article Info

DATE

2005-11-09

AUTHORS

Wolfgang Peti, Rebecca Page, Kin Moy, Mark O’Neil-Johnson, Ian A. Wilson, Raymond C. Stevens, Kurt Wüthrich

ABSTRACT

In structural genomics centers, nuclear magnetic resonance (NMR) screening is in increasing use as a tool to identify folded proteins that are promising targets for three-dimensional structure determination by X-ray crystallography or NMR spectroscopy. The use of 1D 1H NMR spectra or 2D [1H,15N]-correlation spectroscopy (COSY) typically requires milligram quantities of unlabeled or isotope-labeled protein, respectively. Here, we outline ways towards miniaturization of a structural genomics pipeline with NMR screening for folded globular proteins, using a high-density micro-fermentation device and a microcoil NMR probe. The proteins are micro-expressed in unlabeled or isotope-labeled media, purified, and then subjected to 1D 1H NMR and/or 2D [1H,15N]-COSY screening. To demonstrate that the miniaturization is functioning effectively, we processed nine mouse homologue protein targets and compared the results with those from the “macro-scale” Joint Center of Structural Genomics (JCSG) high-throughput pipeline. The results from the two pipelines were comparable, illustrating that the data were not compromised in the miniaturized approach. More... »

PAGES

259-267

References to SciGraph publications

  • 2000-10. Structural proteomics of an archaeon in NATURE STRUCTURAL & MOLECULAR BIOLOGY
  • 1998-07. The second decade — into the third millenium in NATURE STRUCTURAL & MOLECULAR BIOLOGY
  • 2004-03. A scaleable and integrated crystallization pipeline applied to mining the Thermotoga maritima proteome in JOURNAL OF STRUCTURAL AND FUNCTIONAL GENOMICS
  • 2004-09. NMR for structural proteomics of Thermotoga maritima: Screening and structure determination in JOURNAL OF STRUCTURAL AND FUNCTIONAL GENOMICS
  • 2000-11. Protein NMR spectroscopy in structural genomics in NATURE STRUCTURAL & MOLECULAR BIOLOGY
  • 2004-03-08. Fast identification of folded human protein domains expressed in E. coli suitable for structural analysis in BMC MOLECULAR AND CELL BIOLOGY
  • 2000-11. Protein production: feeding the crystallographers and NMR spectroscopists in NATURE STRUCTURAL & MOLECULAR BIOLOGY
  • 2002-09. Role for NMR in structural genomics in JOURNAL OF STRUCTURAL AND FUNCTIONAL GENOMICS
  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1007/s10969-005-9000-x

    DOI

    http://dx.doi.org/10.1007/s10969-005-9000-x

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1000472843

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/16283429


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