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Paper
Mol. BioSyst., 2007, 3, 431 - 438, DOI: 10.1039/b705898p
A novel chemiluminescent substrate for detecting lactamase
Tabassum Naqvi and Rajendra Singh
-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 -lactamase using -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 -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 -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.
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