Towards miniaturization of a structural genomics pipeline using micro-expression and microcoil NMR.
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.
Peti
Wolfgang
W
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.
Page
Rebecca
R
Moy
Kin
K
O'Neil-Johnson
Mark
M
Wilson
Ian A
IA
Stevens
Raymond C
RC
Wüthrich
Kurt
K
D005786:Gene Expression Regulation; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008904:Miniaturization / Q000379:methods*; D015202:Protein Engineering / Q000295:instrumentation / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011506:Proteins