Application of fusion protein 4D5 scFv-dibarnase:barstar-gold complex for studying P185^HER2 receptor distribution in human cancer cells

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• 作者：Julia L. Ivanova^a ; ^{ivajul@mail.ru} ; Evelina F. Edelweiss^b ; Olga G. Leonova^a ; Taras G. Balandin^b ; Vladimir I. Popenko^a ; Sergey M. Deyev^b ; ^c ; ^{deyev@ibch.ru}
• 关键词：P185HER2 ; 4D5 scFv ; Barnase ; Barstar ; Immune electron microscopy
• 刊名：Biochimie
• 出版年：2012
• 期刊代码：24_03009084
• 类别：bio
• 出版时间：August, 2012
• 卷：94
• 期：8
• 页码：1833-1836
• 文件大小：851 K

摘要

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聽掳C and 37聽掳C. It was shown that at 4聽掳C the protein P185^HER2 occurs exclusively on the cell surface, mainly on protrusions or close to their bases. At 37聽掳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.