Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates

Askin, Samuel P., Morin, Isabelle, and Schaeffer, Patrick M. (2011) Development of a protease activity assay using heat-sensitive Tus-GFP fusion protein substrates. Analytical Biochemistry, 415 (2). pp. 126-133.

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Abstract

Proteases are implicated in various diseases and several have been identified as potential drug targets or biomarkers. As a result, protease activity assays that can be performed in high throughput are essential for the screening of inhibitors in drug discovery programs. Here we describe the development of a simple, general method for the characterization of protease activity and its use for inhibitor screening. GFP was genetically fused to a comparatively unstable Tus protein through an interdomain linker containing a specially designed protease site, which can be proteolyzed. When this Tus–GFP fusion protein substrate is proteolyzed it releases GFP, which remains in solution after a short heat denaturation and centrifugation step used to eliminate uncleaved Tus–GFP. Thus, the increase in GFP fluorescence is directly proportional to protease activity. We validated the protease activity assay with three different proteases, i.e., trypsin, caspase 3, and neutrophil elastase, and demonstrated that it can be used to determine protease activity and the effect of inhibitors with small sample volumes in just a few simple steps using a fluorescence plate reader.

Item ID: 17396
Item Type: Article (Research - C1)
ISSN: 1096-0309
Keywords: Tus; Ter; caspase 3; neutrophil elastase; matrix metalloproteinase 13; trypsin; protease activity assay; green fluorescent protein; fusion protein; protein aggregation; denaturation; inhibitor screening
Funders: NHMRC, NIRAP
Projects and Grants: 90030, 90822
Date Deposited: 29 Jun 2011 04:09
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060101 Analytical Biochemistry @ 90%
11 MEDICAL AND HEALTH SCIENCES > 1101 Medical Biochemistry and Metabolomics > 110106 Medical Biochemistry: Proteins and Peptides (incl Medical Proteomics) @ 10%
SEO Codes: 97 EXPANDING KNOWLEDGE > 970103 Expanding Knowledge in the Chemical Sciences @ 100%
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