Application of fusion protein 4D5 scFv-dibarnase:barstar–gold complex for studying P185HER2 receptor distribution in human cancer cells

Overexpression of the P185HER2 protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185HER2 in human cancer cells was studied by electron microscopy, using a novel approach. It is based on the interaction between barnase (a ribonuclease from Bacillus amyloliquefaciens) and its specific inhibitor barstar. The monoclonal antibody 4D5 scFv to extracellular P185HER2 domain fused with two molecules of barnase was used as a recognizing agent, and the conjugate of colloidal gold with barstar, as an electron dense label for electron microscopic visualization. For labeling, we used supramolecular complexes 4D5 scFv-dibarnase:barstar-Au. The distribution of P185HER2 in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was studied at 4 °C and 37 °C. It was shown that at 4 °C the protein P185HER2 occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185HER2 caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185HER2 was located in the coated pits and vesicles, endosomes and multivesicular bodies. The data obtained indicate that the supramolecular 4D5 scFv-dibarnase:barstar–gold complex can be used as a new immunodetection system for exploring the P185HER2 distribution. [Display omitted] ► P185HER2 distribution in cancer cells was studied electron microscopically. ► A novel immunodetection system was used, based on the interaction of barnase with its inhibitor barstar. ► 4D5 scFv fused with two molecules of barnase and barstar-Au was used as labeling complex. ► At 4 °C P185HER2 occurs on the cell surface, at 37 °C its internalization was observed.