A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease.

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.

authors

Cummings RT,Salowe SP,Cunningham BR,Wiltsie J,Park YW,Sonatore LM,Wisniewski D,Douglas CM,Hermes JD,Scolnick EM

doi

10.1073/pnas.062171599

keywords:

subject

Has Abstract

pub_date

2002-05-14 00:00:00

pages

6603-6

issue

10

eissn

0027-8424

issn

1091-6490

pii

062171599

journal_volume

99

pub_type

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