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

J Struct Funct Genomics. 2005 Dec;6(4):259-67. doi: 10.1007/s10969-005-9000-x. Epub 2005 Nov 9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Expression Regulation
  • Magnetic Resonance Spectroscopy / methods*
  • Miniaturization / methods*
  • Protein Engineering / instrumentation
  • Protein Engineering / methods*
  • Proteins / genetics
  • Proteins / isolation & purification
  • Proteins / metabolism

Substances

  • Proteins