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 Lettjournal_title
FEBS lettersauthors
Guignard L,Ozawa K,Pursglove SE,Otting G,Dixon NEdoi
10.1016/s0014-5793(02)03048-xkeywords:
subject
Has Abstractpub_date
2002-07-31 00:00:00pages
159-62issue
1-3eissn
0014-5793issn
1873-3468pii
S001457930203048Xjournal_volume
524pub_type
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