Manoilov K. Biological features of recombinant fragments of diphtheria toxin molecule.

Object: molecular mechanism for implementation of biological activity of non-toxic analogs of diphtheria toxin molecule. Aim: determination of features of the interaction of recombinant derivatives of diphtheria toxin molecule synthesized in Escherichia coli cells with mammalian cells at stages of receptor binding, lipid membrane interactions and endosomal internalization. Methods: general biochemical: electrophoresis in polyacrylamide gel with sodium dodecyl sulfate for analysis of protein samples, electrophoresis of nucleic acids in agarose gel, labeling of proteins with fluoresceinisothiocyanate; chromatographic: metal affinity chromatography for purification of proteins with polygistidine tag; microbiological: cultivation of bacterial strains-producers on cultural media; biotechnological: transformation of prokaryotic cells by vector constructions, cloning of cDNA fragments in plasmid vectors; fluorimetric: flow cytometry, confocal microscopy; in vitro cultures of eukaryotic cells. It was shown for the first time that in contrast to the native diphtheria toxin, its recombinant analogs interact more effectively with receptor of toxin-resistant species and the binding constants of studied compounds and receptors from resistant and cell toxin-sensitive cells were found to be of the same order of magnitude. It has been shown that translocation domain in recombinant derivatives of diphtheria toxin may affect internalization and endosomal transport in cells, retain the inherent toxin ion-conducting function, and involved in the implementation of antitumor activity. The results can be used in development of new oncotherapy technologies.