There is an ongoing development of a novel approach for DNA interaction partners discovery, which is founded on a three-component system that enables enzyme-catalyzed proximity labeling of proteins. The three-component system is comprised of a fusion protein with biotin protein ligase, control fusion protein, and F-DNA that carries DNA fragment of interest and enables colocalization of all described components. Biotin protein ligases are very specific enzymes, but with some mutations applied they become very useful for nonspecific biotinylation of surrounding proteins. That is why they are so useful for protein interaction partners discovery. Up to now all research on the three-component system was made by biotin ligase TurboID. But a big rate of nonspecific biotinylation disturbed the experiment and we became interested if using BioID2 would give a better ratio signal/background. Therefore we planned our experiment so that we would start it with cloning the vector that carries a sequence of BioID2 fusion protein with Tus that binds F-DNA, to continue with expressing recombinant fusion protein to use it in a three-component system. At last, we intended to compare the results of BioID2 and TurboID biotinylation. We succeeded to multiply fragments of a fusion protein with BioID2, assemble them in insert Tus-link72-BioID2, and ligate the insert with a cloning vector to form vector pET-22b(+)_Tus-BioID2 that could be used for fusion protein preparation in bacterial expression system. Afterward, we performed a bioinformatic analysis of TurboID and BioID2. We wanted to additionally explain the mechanism causing faster biotinylation of mutated TurboID and BioID2. That is why we aligned model structures of BioID2 and TurboID with experimentally determined structures of wild-type biotin ligases. We figured out that BioID2 catalytic activity differs from wild-type biotin ligase activity only because of amino acid residue substitution. Small and uncharged glycine stabilizes reactive intermediate in a different way than arginine. We also concluded that modeling the structure of TurboID is not a sufficient way to show the differences in TurboID structure which would provide us with a better understanding of the mechanism for biotinylation acceleration. For more accurate analysis a crystal structure of TurboID should be determined and then compared with a structure of BirA. In the last part, we made a theoretical study of BioID2 and TurboID amino acid structure and defined the next phases that are necessary for our hypothesis verification or disproval.
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