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NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach

Guignard, Laurent; Ozawa, Kiyoshi; Pursglove, Sharon; Dixon, Nicholas; Otting, Gottfried


A cell-free protein expression system was established that provides protein samples of adequate concentration and purity for direct NMR analysis. The Escherichia coli peptidyl-prolyl cis-trans isomerase PpiB was expressed in this system with dual amino acid-selective isotope labeling to identify the NMR signal from the active site-residue Arg87. Addition of the substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide selectively shifted its15N-HSQC cross peak, confirming binding to the active site. As...[Show more]

CollectionsANU Research Publications
Date published: 2002
Type: Journal article
Source: FEBS Letters
DOI: 10.1016/S0014-5793(02)03048-X


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