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A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

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...

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Detalles Bibliográficos
Autores principales: Wigdal, Susan S, Anderson, Jessica L, Vidugiris, Gediminas J, Shultz, John, Wood, Keith V, Fan, Frank
Formato: Texto
Lenguaje:English
Publicado: Bentham Open 2008
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803436/
https://www.ncbi.nlm.nih.gov/pubmed/20161840
http://dx.doi.org/10.2174/1875397300802010016
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author Wigdal, Susan S
Anderson, Jessica L
Vidugiris, Gediminas J
Shultz, John
Wood, Keith V
Fan, Frank
author_facet Wigdal, Susan S
Anderson, Jessica L
Vidugiris, Gediminas J
Shultz, John
Wood, Keith V
Fan, Frank
author_sort Wigdal, Susan S
collection PubMed
description 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.
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spelling pubmed-28034362010-02-16 A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase Wigdal, Susan S Anderson, Jessica L Vidugiris, Gediminas J Shultz, John Wood, Keith V Fan, Frank Curr Chem Genomics Article 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. Bentham Open 2008-10-17 /pmc/articles/PMC2803436/ /pubmed/20161840 http://dx.doi.org/10.2174/1875397300802010016 Text en © Wigdal et al.; Licensee Bentham Open. http://creativecommons.org/licenses/by-nc/3.0/ 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.
spellingShingle Article
Wigdal, Susan S
Anderson, Jessica L
Vidugiris, Gediminas J
Shultz, John
Wood, Keith V
Fan, Frank
A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title_full A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title_fullStr A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title_full_unstemmed A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title_short A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase
title_sort novel bioluminescent protease assay using engineered firefly luciferase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2803436/
https://www.ncbi.nlm.nih.gov/pubmed/20161840
http://dx.doi.org/10.2174/1875397300802010016
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