Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.

中文翻译：

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基于二聚化的红色荧光蛋白的蛋白酶生物传感器的开发

蛋白酶Matriptase的活性失调是侵袭性肿瘤生长，癌症转移和骨关节炎的关键因素。检测和定量苹果酸酶活性和抑制的方法将是有用的工具。为了满足这一需求，我们开发了一种基于二聚体依赖性红色荧光蛋白（ddRFP）报告系统的麦芽糖酶敏感蛋白生物传感器。在这个平台上，通过蛋白酶不稳定的连接子连接的两个相邻的蛋白结构域在组装时产生荧光，而当该连接子被麦芽糖酶裂解时则不发光。创建了一组基于ddRFP的Matriptase生物传感器设计，该设计在蛋白质结构域之间包含不同的接头长度。这些构建体的特征在于接头特异性切割，matriptase活性和matriptase选择性。包含RSKLRVGGH接头（称为B4）的生物传感器以高收率表达，并显示出高催化效率和Matriptase特异性。该生物传感器可检测可溶性和酵母细胞表面表达的抑制剂域（最高可达5倍动态范围）对脂蛋白酶的抑制作用，还可检测人癌细胞系表达的脂蛋白酶活性。除苹果酸酶外，我们重点介绍了可用于创建有效的生物传感器以量化活性和抑制其他目的蛋白酶的策略。该生物传感器可检测可溶性和酵母细胞表面表达的抑制剂域（最高可达5倍动态范围）对脂蛋白酶的抑制作用，还可检测人癌细胞系表达的脂蛋白酶活性。除苹果酸酶外，我们重点介绍了可用于创建有效的生物传感器以量化活性和抑制其他目的蛋白酶的策略。该生物传感器可检测可溶性和酵母细胞表面表达的抑制剂域（最高可达5倍动态范围）对脂蛋白酶的抑制作用，还可检测人癌细胞系表达的脂蛋白酶活性。除苹果酸酶外，我们重点介绍了可用于创建有效的生物传感器以量化活性和抑制其他目的蛋白酶的策略。