State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
Talanta. 2013 May 15;109:141-6. doi: 10.1016/j.talanta.2013.02.007. Epub 2013 Feb 8.
Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:~110 nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1 pM in pure buffer and 3.3 pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.
由于介质复杂，在真实生理环境中监测蛋白酶仍然是一个巨大的挑战。生物发光共振能量转移（BRET）是一种很有前途的技术，但它的应用受到缓冲液组成的影响，这可能会导致检测出现严重误差。在此，我们报告了一种将 BRET 对与人源化海肾荧光素酶（hGluc）和高亮度红色荧光蛋白 tdTomato 相结合的新方法，用于灵敏和稳健的蛋白酶活性测定。结果表明，hGluc/tdTomato BRET 对缓冲液组成、pH 值和样品基质具有更好的耐受性，且光谱分离度宽（Δλ：~110nm）。利用该对构建的蛋白酶传感器，在纯缓冲液中检测肠激酶的检测限达到 2.1pM，在 3%血清中为 3.3pM。该对有望在体外和体内检测中得到广泛应用，特别是对于基质复杂的样品。
