A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
Susan S Wigdal, Jessica L Anderson, Gediminas J Vidugiris, John Shultz, Keith V Wood, Frank Fan*
Identifiers and Pagination:Year: 2008
First Page: 16
Last Page: 28
Publisher Id: CCGTM-2-16
Article History:Received Date: 20/5/2008
Revision Received Date: 20/8/2008
Acceptance Date: 23/8/2008
Electronic publication date: 17/10/2008
Collection year: 2008
open-access license: This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.