A peptide-based fluorescence resonance energy transfer assay for Bacillus anthracis lethal factor protease.
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.
Cummings
Richard T
RT
Department of High Throughput Screening and Automation, Merck Research Laboratories, Rahway, NJ 07065-0900, USA. richard_cummings@merck.com
Salowe
Scott P
SP
Cunningham
Barry R
BR
Wiltsie
Judyann
J
Park
Young Whan
YW
Sonatore
Lisa M
LM
Wisniewski
Douglas
D
Douglas
Cameron M
CM
Hermes
Jeffrey D
JD
Scolnick
Edward M
EM
D000942:Antigens, Bacterial; D001408:Bacillus anthracis / Q000201:enzymology*; D001427:Bacterial Toxins / Q000378:metabolism*; D048369:MAP Kinase Kinase 1; D008666:Metalloendopeptidases / Q000378:metabolism*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D010455:Peptides; C030325:anthrax toxin; D017346:Protein Serine-Threonine Kinases; D048369:MAP Kinase Kinase 1; D020929:Mitogen-Activated Protein Kinase Kinases; D008666:Metalloendopeptidases