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

Authors

Laurent Guignard, Australian National University
Kiyoshi Ozawa, University of WollongongFollow
Sharon E. Pursglove, Australian National University
Gottfried Otting, Australian National University
Nicholas E. Dixon, University of WollongongFollow

RIS ID

16965

Publication Details

Guignard, L., Ozawa, K., Pursglove, S. E., Otting, G. & Dixon, N. E. (2002). NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach. FEBS Letters, 524 (1-3), 159-162.

Abstract

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 its ¹⁵N-HSQC cross peak, confirming binding to the active site. As cell-free protein expression provides high yields of protein per unit mass of labeled amino acid and sample handling is minimal, this strategy presents an exceptionally inexpensive and rapid approach to protein analysis.