The histone-like protein of the hyperthermophilic eubacterium Thermotoga maritima is a member of the family of HU proteins. The main function of HU proteins is the condensation or packaging of DNA. The melting point of the TmHU homodimer was determined at 95 °C. Because of its high content of basic amino acids, TmHU is able to interact with DNA. In the context of this work, the suitability of TmHU for gene therapeutic approaches was examined. It was investigated, if TmHU is able to transfer nucleic acids, like plasmid DNA or siRNA, into human cells. In addition to the ability to interact with RNA, TmHU is able to reach the inside of cells, where it is located in the cytoplasm and the nucleus. By western blot analysis, the protein is detectable up to 96 hours in the lysate of human cells. Furthermore, it mediates the transfer of siRNA into human cells. By using the endocytosis inhibitor cytochalasin D, the uptake mechanism of the complexes of TmHU and siRNA was elucidated. The precipitate, which forms during the interaction of siRNA and TmHU, is taken up by endocytosis, followed by a degradation of the complexes of TmHU and siRNA. By addition of chloroquine into the transfection approach, endosomolysis in the investigated cells was caused. About 30 % of the cells expressed EGFP after the transfection of an EGFP plasmid with TmHU. Because of the toxicity of chloroquine, an alternative to destabilise the endosomoes was proven. Thereto, the variant E5 of the fusogenic peptide of hemagglutinin HA-2 of the influenza virus was coupled covalently to TmHU. After transfection of cells with an EGFP plasmid and the product of the coupling reaction TmHU-E5, up to 31 % of the investigated cells expressed EGFP. In combination with chloroquine the transfection rates were even far in excess of 60 %. Because of the remarkable properties of TmHU, an efficient gene transfer system for DNA and RNA into human cells was developed in vitro. Due to its thermostability and its high biotechnological availability, an application of TmHU in the field of gene therapy is conceivable. |