A novel chemiluminescent substrate for detecting lactamase.
Beta-lactamase is a well established reporter for monitoring cellular events while chemiluminescence is the preferred read-out mode in high throughput screens. Here, we report the first chemiluminescent assay for beta-lactamase using beta-galactosidase based enzyme fragment complementation technology. The enzyme fragment complementation technology employs a large protein fragment called the enzyme acceptor and a small peptidic fragment called an enzyme donor. These fragments are inactive separately but recombine rapidly in solution to yield active beta-galactosidase detected by chemiluminescence or fluorescence. A cyclic enzyme donor comprising a substituted cephalosporin moiety is used as the lactamase substrate. The cyclic substrate does not complement with enzyme acceptor to yield active beta-galactosidase, but upon cleavage with lactamase yields the linear enzyme donor which complements readily with enzyme acceptor. This methodology has been exploited in a simple, sensitive, homogeneous cell based reporter gene assay to monitor G-protein coupled receptor activation in a microtitre plate with a chemiluminescent read out.
Naqvi
Tabassum
T
DiscoveRx Corporation, 42501 Albrae Street, Fremont, CA 94538, USA.
Singh
Rajendra
R
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003500:Cyclization; D004926:Escherichia coli / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D010539:Permeability; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D001618:beta-Lactamases