| Affiliation: | (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;(2) Sequoia Sciences, Inc., 11199 Sorrento Valley Road, San Diego, CA 92121, USA;(3) Present address: Department of Molecular Pharmacology, Physiology and Biotechnology, 70 Ship Street, Providence, RI 02912, USA;(4) Present address: Brown Universuty, Brown University, Department of Molecular Biology, Cell Biology and Biochemistry, 70 Ship Street, Providence, RI 02912, 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. |
