Title

15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes

RIS ID

16847

Publication Details

Ozawa, K., Wu, P. S.C., Dixon, N. E. & Otting, G. (2006). 15N-Labelled proteins by cell-free protein synthesis. Strategies for high-throughput NMR studies of proteins and protein-ligand complexes. The FEBS Journal, 273 (18), 4154-4159.

Abstract

Abstract

[15N]-heteronuclear single quantum coherence (HSQC) spectra provide a readily accessible fingerprint of [15N]-labelled proteins, where the backbone amide group of each nonproline amino acid residue contributes a single cross-peak. Cell-free protein synthesis offers a fast and economical route to enhance the information content of [15N]-HSQC spectra by amino acid type selective [15N]-labelling. The samples can be measured without chromatographic protein purification, dilution of isotopes by transaminase activities are suppressed, and a combinatorial isotope labelling scheme can be adopted that combines reduced spectral overlap with a minimum number of samples for the identification of all [15N]-HSQC cross-peaks by amino acid residue type. These techniques are particularly powerful for tracking [15N]-HSQC cross-peaks after titration with unlabelled ligand molecules or macromolecular binding partners. In particular, combinatorial isotope labelling can provide complete cross-peak identification by amino acid type in 24 h, including protein production and NMR measurement.

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Link to publisher version (DOI)

http://dx.doi.org/10.1111/j.1742-4658.2006.05433.x