Department of Life Science, Hanyang University, Seoul 04763, Korea.
Brain Korea 21 Plus BioDefense Research Team, Hanyang University, Seoul 04763, Korea.
Sensors (Basel). 2018 Mar 15;18(3):875. doi: 10.3390/s18030875.
We report bioluminescence analysis of matrix metalloproteinase (MMP) activity in biological substances using a surface-bound luciferase probe. Intein-fused luciferase protein enables site-specific biotinylation of luciferase in the presence of N-terminus cysteine-biotin via intein-mediated splicing process, resulting in a strong association with high bioluminescence signal onto a NeutrAvidin-coated surface. When the peptide substrate for MMP-7 was inserted into a region between luciferase and intein, the biotinylated probe detected MMP-7 activity by cleaving the peptide, and surface-induced bioluminescence signal was strongly reduced in the MMP-secreted media or mouse tissue extracts, compared with that in MMP-deficient control set. Our approach is anticipated to be useful for generating biotinylated proteins and for their applications in diagnosing MMP activity in human diseases.
我们报告了使用表面结合的荧光素酶探针对生物物质中基质金属蛋白酶(MMP)活性进行生物发光分析。融合了内含肽的荧光素酶蛋白在 N 端半胱氨酸-生物素的存在下,通过内含肽介导的剪接过程,实现了荧光素酶的定点生物素化,从而与链霉亲和素涂层表面强烈结合,产生强的生物发光信号。当 MMP-7 的肽底物插入到荧光素酶和内含肽之间的区域时,生物素化探针通过切割肽检测 MMP-7 的活性,与 MMP 缺陷对照组相比,在 MMP 分泌的介质或小鼠组织提取物中,表面诱导的生物发光信号显著降低。我们的方法有望用于生成生物素化蛋白,并将其应用于诊断人类疾病中的 MMP 活性。