Understanding the interactions between small molecules and macromolecules from different perspectives is important for the advancement of basic science and drug development. Towards this goal, we usually begin with a known macromolecule and discover small molecules that interact with it. Alternatively, looking for macromolecules that interact with a selected small molecule, whose biological (phenotypic) activity may or may not be known. Affinity chromatography is the most widely used technique for isolating specific target proteins from a complex proteome. The aim of the master's thesis was to identify proteins that specifically interact with two similar small molecules, 4- (2-aminoethyl) -1-cyclohexyl pyrazol-5-ol (ligand 1) and 4- (2-aminoethyl)-1-phenyl pyrazole-5-ol (ligand 2), which were synthesized at the Department of Organic Chemistry UL FCCT. Both molecules were individually immobilized on NHS-activated Sepharose™ 4 Fast flow agarose and the prepared columns were used to isolate target proteins. The test sample was a lysate of U937 human monocyte cells cultured in suspension. SDS-PAGE analyzes showed that the ligands differed from each other in the set of interacting proteins. Eluates from the ligand 1 column were sent for mass spectrometry analysis, thus identifying one of the interacting proteins as ISOC2, a little-known human protein that has an isochorimatase domain and interacts with the tumor inhibitor p16INK4a. If ISOC2 were a regulator of p16INK4a, it might be a new target for development of a novel strategy for the treatment of cancer and aging. For the purpose of more detailed characterization of the in vitro interaction, we developed a system for expressing the human ISOC2 form in E. coli. Analysis of the recombinant protein by size separation chromatography showed that it was most likely a homodimer, distinguishing it from its homologues, which are mostly homotetramers.
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