Production and development of fluorescent protein based fret biosensor

Förster or Fluorescent Resonance Energy Transfer (FRET) is defined as non-radioactive energy transfer between two fluorophores. Three main types of fluorophores are used in FRET biosensors, small organic dyes, fluorescent proteins and quantum dots. Fluorescent proteins are widely used because they are genetically coding and compatible with cells. Fluorescent Protein-based FRET (FP-FRET) biosensors are used to display biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. With the presented thesis study, it was aimed to determine the protease activity with the help of the FP-FRET biosensor. In this context, TEV protease enzyme was chosen as a model. Plasmids of FRET biosensors containing mClover3-mRuby3 and mNeonGreen-mRuby3 fluorescent pairs were designed. Expression of the gene of the designed FP-FRET biosensor was performed in E. coli DH5α cells using recombinant DNA techniques. The expressed fusion protein was purified by affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed on the spectrofluorometer device using the FP-FRET biosensor produced. In later studies, it is aimed to produce new FP-FRET biosensors for the activity determination of enzymes used in important biological processes.