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

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 (15)N-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.

journal_name

FEBS Lett

journal_title

FEBS letters

authors

Guignard L,Ozawa K,Pursglove SE,Otting G,Dixon NE

doi

10.1016/s0014-5793(02)03048-x

keywords:

subject

Has Abstract

pub_date

2002-07-31 00:00:00

pages

159-62

issue

1-3

eissn

0014-5793

issn

1873-3468

pii

S001457930203048X

journal_volume

524

pub_type

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