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

Abstract
Overexpression of the P185(HER2) protein determines the malignancy and unfavorable prognosis of ovarian and breast tumors. In this work, the distribution of P185(HER2) 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 P185(HER2) 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 P185(HER2) 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 P185(HER2) occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37 °C, the internalization of P185(HER2) caused by its interaction with 4D5 scFv-dibarnase was observed. Inside the cells, P185(HER2) 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 P185(HER2) distribution.
AuthorsJulia L Ivanova, Evelina F Edelweiss, Olga G Leonova, Taras G Balandin, Vladimir I Popenko, Sergey M Deyev
JournalBiochimie (Biochimie) Vol. 94 Issue 8 Pg. 1833-6 (Aug 2012) ISSN: 1638-6183 [Electronic] France
PMID22531627 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2012 Elsevier Masson SAS. All rights reserved.
Chemical References
  • Antibodies, Monoclonal
  • Bacterial Proteins
  • Recombinant Fusion Proteins
  • immunoRNase scFv 4D5-dibarnase
  • barstar protein, Bacillus amyloliquefaciens
  • Gold
  • ERBB2 protein, human
  • Receptor, ErbB-2
  • Ribonucleases
Topics
  • Antibodies, Monoclonal (chemistry)
  • Bacterial Proteins (chemistry)
  • Breast Neoplasms (metabolism)
  • Cell Line, Tumor
  • Coated Pits, Cell-Membrane (ultrastructure)
  • Coated Vesicles (ultrastructure)
  • Endosomes (ultrastructure)
  • Female
  • Gold (chemistry)
  • Humans
  • Multivesicular Bodies (ultrastructure)
  • Ovarian Neoplasms (metabolism)
  • Receptor, ErbB-2 (analysis, metabolism)
  • Recombinant Fusion Proteins (chemistry, immunology)
  • Ribonucleases (chemistry, immunology)
  • Staining and Labeling
  • Temperature

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