A novel method to detect and measure protease activities using a genetically engineered firefly (Photinus pyralis) luciferase, representing one example of the GloSensor™ platform technology. The Protease-Glo™ Assay can be used to:
- Measure specificity of proteases or protease recognition sequences.
- Compare related proteases against the same substrate (e.g., viral strains).
- Determine protease substrate specificity (length of essential sequence, what specific amino acids are essential).
- Screen for protease inhibitors and determine inhibitor potencies (IC50).
The assay uses a circularly permuted firefly luciferase, the GloSensor™-10F protein, with a protease recognition site as the protease substrate. This assay system allows rapid generation of protease substrates through molecular cloning and coupled transcription/translation cell-free expression, thus enabling the facile evaluation of protease function. Oligonucleotides encoding a protease recognition sequence are designed and cloned into the GloSensor™-10F gene located on a linearized vector. The GloSensor™ protein containing the protease site of interest is then synthesized in a cell-free protein expression system and subsequently used as a protease substrate. Cleavage of the protease recognition sequence leads to activation of the GloSensor™ protein and light emission. The level of luminescence correlates to protease activity. The Protease-Glo™ Assay has the advantage of a bioluminescent readout, which provides easy quantitation, high sensitivity and wide dynamic range. The physical and chemical features of luminescence overcome problems due to fluorescence interference.
- Fan, F. et al. (2008) Novel genetically encoded biosensors using firefly luciferase. ACS Chem. Biol. 3, 346–51.
- Wigdal, S. et al. (2008) A novel bioluminescent protease assay using engineered firefly luciferase. Curr. Chem. Genomics 1, 94–106.
- Fan, F. and Wood, K.V. (2007) Bioluminescent assays for high-throughput screening. Assay Drug. Dev. Technol. 5, 127–136.