Comparative Genomics Centre, School of Pharmacy and Molecular Sciences, James Cook University, DB 21, James Cook Drive, Townsville, QLD 4811, Australia.
Anal Biochem. 2011 Aug 15;415(2):126-33. doi: 10.1016/j.ab.2011.04.028. Epub 2011 Apr 27.
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.
蛋白酶与多种疾病有关，其中一些已被确定为潜在的药物靶点或生物标志物。因此，能够进行高通量筛选的蛋白酶活性测定对于药物发现计划中的抑制剂筛选至关重要。在这里，我们描述了一种简单、通用的蛋白酶活性分析方法的开发及其在抑制剂筛选中的应用。GFP 通过含有专门设计的蛋白酶切割位点的结构域间连接子与相对不稳定的 Tus 蛋白发生基因融合，该切割位点可被蛋白酶切割。当 Tus-GFP 融合蛋白底物被蛋白酶切割时，GFP 被释放出来，在短暂的热变性和离心步骤后，GFP 仍留在溶液中，该步骤用于去除未切割的 Tus-GFP。因此，GFP 荧光的增加与蛋白酶活性直接成正比。我们使用三种不同的蛋白酶（即胰蛋白酶、caspase 3 和中性粒细胞弹性蛋白酶）验证了蛋白酶活性测定法，并证明它可以用于在使用荧光板读数器的短短几个简单步骤中，使用少量样品体积确定蛋白酶活性和抑制剂的效果。
