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NMR analysis of in vitro‐synthesized proteins without purification: a high‐throughput approach
Author(s) -
Guignard Laurent,
Ozawa Kiyoshi,
Pursglove Sharon E,
Otting Gottfried,
Dixon Nicholas E
Publication year - 2002
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(02)03048-x
Subject(s) - heteronuclear single quantum coherence spectroscopy , escherichia coli , cell free protein synthesis , amino acid , biochemistry , prolyl isomerase , substrate (aquarium) , residue (chemistry) , chemistry , in vitro , stable isotope labeling by amino acids in cell culture , isomerase , biology , nuclear magnetic resonance spectroscopy , protein biosynthesis , stereochemistry , enzyme , proteomics , ecology , gene , pin1
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.