Genetically engineered
bacterial protein toxins are attractive systems for delivery of exogenous
proteins into the cytosol of mammalian cells. The binary C2 toxin from C. botulinum has emerged as powerful delivery vehicle, which rests on its binding/translocation component C2IIa and the genetically modified adaptor domain C2IN that act in concert to trigger cell uptake. The
p53 tumor suppressor protein has a crucial function in suppressing
carcinogenesis and is frequently inactivated by diverse mechanisms in human
tumor cells. Therefore, we constructed a C2IN-p53 fusion
protein, which is internalized into
cancer cells by C2IIa. To this end, the C2IN-p53 fusion construct was overexpressed in E. coli with good solubility, purified by
heparin affinity chromatography and
protein identity was confirmed by immunoblotting. We demonstrated that the fusion
protein is capable of binding to the p53 consensus-
DNA with high affinity in a p53-specific manner in vitro. Next, the internalization of C2IN-p53 was monitored in HeLa cells by cell fractionation and immunoblot analysis, which revealed a C2IIa-mediated translocation of the fusion
protein into the cytosol. The uptake was also shown in A549 and Saos-2 cells with similar efficiency. These findings were further corroborated by confocal immunofluorescence analyses of C2IN-p53/C2IIa-treated HeLa and A549 cells, displaying predominantly cytoplasmic localization of the fusion construct.