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Journal of Structural and Functional Genomics

, Volume 6, Issue 4, pp 259–267 | Cite as

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

  • Wolfgang Peti
  • Rebecca Page
  • Kin Moy
  • Mark O’Neil-Johnson
  • Ian A. Wilson
  • Raymond C. StevensEmail author
  • Kurt Wüthrich
Article

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.

Keywords

microcoil NMR micro-expression NMR screening structural genomics 

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Notes

Acknowledgements

The JCSG consortium is funded by NIH GM062411. W.P. is a Max Kade Foundation fellow, and K.W. is the Cecil H. and Ida M. Green Professor of Structural Biology at TSRI. The development of the microcoil NMR probe was in part financed by the NIH SBIR RR15384, and we also thank Spectra Stable Isotopes, Cambridge Isotope Laboratories and MRM/Protasis for support.

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Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • Wolfgang Peti
    • 1
    • 3
  • Rebecca Page
    • 1
    • 4
  • Kin Moy
    • 1
  • Mark O’Neil-Johnson
    • 2
  • Ian A. Wilson
    • 1
  • Raymond C. Stevens
    • 1
    Email author
  • Kurt Wüthrich
    • 1
  1. 1.The Scripps Research InstituteDepartment of Molecular Biology and the Joint Center of Structural GenomicsLa JollaUSA
  2. 2.Sequoia Sciences, Inc.San DiegoUSA
  3. 3.Department of Molecular Pharmacology, Physiology and BiotechnologyProvidenceUSA
  4. 4.Brown UniversutyBrown University, Department of Molecular Biology, Cell Biology and BiochemistryProvidenceUSA

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