| Literature DB >> 11997440 |
Richard T Cummings1, Scott P Salowe, Barry R Cunningham, Judyann Wiltsie, Young Whan Park, Lisa M Sonatore, Douglas Wisniewski, Cameron M Douglas, Jeffrey D Hermes, Edward M Scolnick.
Abstract
A fluorescence resonance energy transfer assay has been developed for monitoring Bacillus anthracis lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9-16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH(2), was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.Entities:
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Year: 2002 PMID： 11997440 PMCID： PMC124449 DOI： 10.1073/pnas.062171599
Source DB: PubMed Journal: Proc Natl Acad Sci U S A ISSN： 0027-8424 Impact factor: 11.205