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

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Journal of Structural and Functional Genomics

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.

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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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Author notes

  1. Wolfgang Peti

    Present address: Department of Molecular Pharmacology, Physiology and Biotechnology, 70 Ship Street, Providence, RI, 02912, USA

  2. Rebecca Page

    Present address: Brown Universuty, Brown University, Department of Molecular Biology, Cell Biology and Biochemistry, 70 Ship Street, Providence, RI, 02912, USA

Authors and Affiliations

  1. The Scripps Research Institute, Department of Molecular Biology and the Joint Center of Structural Genomics, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA

    Wolfgang Peti, Rebecca Page, Kin Moy, Ian A. Wilson, Raymond C. Stevens & Kurt Wüthrich

  2. Sequoia Sciences, Inc., 11199 Sorrento Valley Road, San Diego, CA, 92121, USA

    Mark O’Neil-Johnson

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  1. Wolfgang Peti
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  2. Rebecca Page
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  4. Mark O’Neil-Johnson
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  5. Ian A. Wilson
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  6. Raymond C. Stevens
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  7. Kurt Wüthrich
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Corresponding author

Correspondence to Raymond C. Stevens.

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Peti, W., Page, R., Moy, K. et al. Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR. J Struct Funct Genomics 6, 259–267 (2005). https://doi.org/10.1007/s10969-005-9000-x

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  • DOI: https://doi.org/10.1007/s10969-005-9000-x

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