İbrahim İNCİR, İsa GÖKÇE, Özlem KAPLAN, Sema BİLGİN

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

Proteases are closely associated with many pathological conditions. Efficient detection ofprotease activity may be useful for diagnosis, prognosis, and the development of newtherapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as thenon-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins arewidely used in FRET biosensors because they can be genetically encoded and compatible withcells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biologicalprocesses such as enzyme activity, intracellular ion concentration, conformational changes,protein-protein interactions. In this study, it was aimed to detect protease activity using an FPFRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding themNeonGreen-TEV-mRuby3 fluorescent protein-based FRET biosensor was constructed. Thegene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells usingrecombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result,the activity of the TEV protease enzyme was determined by emission measurements performedin the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designedFP-FRET biosensor in the determination of protease enzyme activity was demonstrated.

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