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

被引：61

作者：

Guignard, L

Ozawa, K

Pursglove, SE

Otting, G

Dixon, NE ^{[1
]}

机构：

[1] Australian Natl Univ, Res Sch Chem, Canberra, ACT 0200, Australia

[2] Karolinska Inst, Dept Med Biochem & Biophys, S-17177 Stockholm, Sweden

来源：

FEBS LETTERS | 2002年 / 524卷 / 1-3期

关键词：

in vitro protein expression; cell-free; high throughput; PpiB; prolyl cis-trans isomerase;

D O I：

10.1016/S0014-5793(02)03048-X

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 N-15-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. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

引用

页码：159 / 162

页数：4

共 26 条

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