P Lactamase

Bacterial P-lactamase is another enzyme with characteristics desirable for use as reporter gene42 and for which colorimetric substrates have been known for a long time. The P-lactamase cell-based assay system couples molecular and biological events to a fluorescence resonance energy transfer (FRET)-based detection method in single live cells (GeneBLAzerâ„¢ technology, Invitrogen). Since there is no mammalian counterpart, its expression is background-free. Its half-life is similar to that of beetle luciferase. The development of a membrane-permeable, self-quenched, fluorescence resonance energy-transfer substrate a few years ago allows visualization of enzyme activity in living cells43 and also delivers a ratiometric readout. The substrate consists of a fluores-cein moiety as the acceptor fluorophore attached to a coumarin moiety, which is the fluorescence donor. Excitation of the donor at 409 nm leads to emission of green light (529 nm) from the uncleaved substrate. P-lactamase cleavage releases fluorescein and leads to the emission of blue light (447 nm). The ratio of the emission at 447 nm to that at 520 nm is a concentration-independent measure of the extent of the reaction. An esterified derivative of the substrate is non-fluorescent and only metabolizes to the fluorescent version when cleaved by cytoplasmic esterases in the cell. The high sensitivity of the assay, which requires only 100 reporter molecules per cell, allows flourescene-activated cell sorter (FACS) analysis of cell suspensions and clonal selection of transfected cells, thus significantly shortening the time to genetically engineer the reporter cell line. The ratiometric readout normalizes the signal to cell number, which is especially useful for compound screening. As described before, it can correct for edge effects, which can occur during longer incubation times of plated cells and for cells of different viability. These characteristics together with the very high sensitivity of the differential assay make it amenable to remarkable miniaturization beyond the

384-3456-well plate format.44 The properties described before make the P-lactamase a very attractive reporter gene, especially for screening purposes, since edge effects and toxic effects of test compounds can significantly impact the test results. However, utilizing the P-lac reporter system, we observed that the substrate CCF2-AM exhibits a general instability in aqueous solution increasing the background fluorescence and resulting in a non-P-lac-induced conversion of the substrate in the cell, which leads to an increase of the blue fluorescence even in the absence of P-lac. However, this effect depends very much on the cell type used for the assay.

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