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J Struct Funct Genomics. 2005 Dec;6(4):259-67. Epub 2005 Nov 9.

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

Author information

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.

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.

PMID:
16283429
DOI:
10.1007/s10969-005-9000-x
[Indexed for MEDLINE]

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