Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein

Abstract

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.

Biosensors for Detection of Biochemical Markers Relevant to Osteoarthritis

“…Studies were published between 2003 [ 12 ] and 2020 [ 2 , 33 ]. Biomarkers were found in the synovial fluid [ 8 , 12 , 31 , 32 , 34 , 35 ] in serum and urine [ 2 , 4 , 5 , 10 , 14 , 36 , 37 ]; in blood and uCTX-II control [ 8 , 22 , 38 ] in DNA extracted; in epithelial cells and in bovine articular cartilage [ 24 , 33 , 39 ].…”

“…The most common biosensors adopted were FOPPR [ 31 , 32 , 35 ], SPRi and FMGC biosensors [ 8 , 10 , 12 , 36 ]. Other biosensors used were MIP, Quartz crystal microbalance biosensor, AMPK, FBG, anti-COMP, DdRFP, FCD, IDE, QCM, SAM, hand-held optical biosensing system utilizing a smartphone-embedded illumination sensor that is integrated with the immunoblotting assay method and amperometric biosensor [ 2 , 4 , 5 , 10 , 14 , 22 , 24 , 25 , 33 , 34 , 37 , 39 ].…”

“…The most common biomarkers were CTX-II, both uCTX-II and sCTX-II [ 2 , 10 , 14 , 22 , 37 , 38 ], followed by COMP, MMP-1 and MMP-3 [ 4 , 5 , 8 , 31 , 32 , 34 ]. Other biomarkers adopted were: CRP, GPI, TNF, ECM, Mitochondrial DNA, Interleukin-1B, Protease matriptase and Uricase enzyme layer thickness [ 12 , 24 , 25 , 33 , 35 , 36 , 39 ].…”

“…The QCM biosensors could detect the mass change that occurs during the binding process [ 5 ]. Molecular biosensors are devices that can measure biological processes, such as protein–protein interactions, cell and molecular trafficking or protease activity, through a signal readout [ 24 , 25 ].…”

Current and Emerging Approaches for Studying Inter-Organelle Membrane Contact Sites

“…The ER-mitochondria MCS was allowed for detection and monitored by using this sensor in HeLa and HEK293 cells (Cieri et al, 2018). ddFP Dimerization-dependent fluorescent protein (ddFP) is a class of genetically encoded reporters based on the reversible binding of two dark fluorescent protein monomers to form a fluorescent heterodimeric complex, which can be used for detection of protein-protein interactions in living cells (Ding et al, 2015;Mitchell et al, 2018). The yellow or red fluorescent proteins are obligate tetrameric, however, is limited by its tetramerization disruption.…”