A. Niles1, C. Brouette1, R. Moravec1, M. Scurria2, B. Daily2, F. Fan1, B. Butler1, K. Lewis1, S. Frackman1, F. Huang1, K. Wood1 and T. Riss1
1Promega Corporation, 2800 Woods Hollow Road, Madison WI 53711
2Promega Biosciences, 277 Granada Drive, San Luis Obispo, CA 93401
Bioluminescence has gained favor in recent years as a detection technology in the drug discovery process because the chemistry offers large signal windows while exhibiting extremely low intrinsic backgrounds. These attributes allow for high assay sensitivity and an extended linear range when compared to similar fluorescence measures. We have developed two distinct bioluminescent platforms useful for measuring the diversity of activities represented by the major protease classes. The first assay format utilizes a circularly permuted firefly luciferase, artificially joined at the original enzyme termini by cloning in a protease peptide recognition sequence of interest. In the presence of a specific protease, luciferin, ATP, and MgSO4, the luciferase enzyme demonstrates enhanced activity and generates a light signal proportional to protease activity. In the absence of specific protease activity, the engineered luciferase demonstrates poor luciferin catalysis and low light output. This platform is broadly applicable to all proteases but represents particular utility with metallo- and aspartic class proteases, which typically require both P and P′ amino acids in their recognition sites. An additional feature of this molecular approach is that it offers facile analysis of substrate specificity without chemical synthesis of peptides. The second assay utilizes protease-selective peptide-derivatized aminoluciferin substrates, which are introduced into a test sample with a thermostable beetle luciferase, ATP, and MgSO4. In the presence of specific endoproteolytic activity, cleavage is mediated at the scissile site between the P1 amino acid and aminoluciferin, liberating the substrate for luciferase and generating a light signal proportional to the protease activity. In the absence of specific protease activity, the peptide-derivatized aminoluciferin substrate is a poor substrate for luciferase and generates minimal light. This platform is amenable to members of the serine and cysteine classes. Together, these novel platforms allow for sensitive and robust measurement of therapeutically relevant proteolytic activities in both biochemical and cell-based assay formats.