Morozova K. Structural and functional properties of the far-red fluorescent protein TagRFP657 and cell biology application.

The object of the research: far-red fluorescent protein TagRFP657 and it's mutant variants. The objective of the theses: as original template for the molecular evolution we used a FP mKate with a fluorescence emission peak at 635 nm to design new far-red fluorescent variant TagRFP657 with a fluorescence emission peak above 650 nm. To determine the connection between structure of TagRFP657 and its fluorescent and biochemical properties and discover the possibilities of cell biology applications of TagRFP657. The research methods: polymerase chane reaction, electrophoresis in polyacrylamide gel, mammalian cell culture and transfection, random and site-directed mutagenesis, sequencing, spectrophotometry, flow cytometry, fluorescent microscopy. The results, novelty: the theses presents the new fluorescent protein TagRFP657 described by an excitation peak at 611 nm and a fluorescence emission peak at 657 nm. A recombinant purified TagRFP657 has a pH-stability with an apparent рКа value of 5,0. An extinction coefficient of RFP657 is 34000 М-1.см-1, and its quantum yield is 0,10 and photobleaching half-time is 55 sec. The chromophore maturation half-time of TagRFP657 is 125 min. In model experiments TagRFP657 was successfully expressed and visualized untagged and in fusions in mammalian cells. Far-red fluorescent protein TagRFP657 will expand possibilities of flow cytometry applications. The sphere of application: cell biology, oncology, pharmacology, immunology.