NMR analysis of in vitro-synthesized proteins without purification: a high-throughput approach.
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.
Guignard
Laurent
L
Research School of Chemistry, Australian National University, Canberra, ACT, Australia.
Ozawa
Kiyoshi
K
Pursglove
Sharon E
SE
Otting
Gottfried
G
Dixon
Nicholas E
NE
D001226:Aspartate-tRNA Ligase / Q000737:chemistry*; D001331:Automation; D002474:Cell-Free System; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D019696:Peptidylprolyl Isomerase / Q000737:chemistry*
D029968:Escherichia coli Proteins; D007527:Isoenzymes; C421208:peptidyl-prolyl cis-trans isomerase b; D019696:Peptidylprolyl Isomerase; D001226:Aspartate-tRNA Ligase