Affinity-based entrapment of the HER2 receptor in the endoplasmic reticulum using an affibody molecule

• Interference with the export of cell surface receptors can be performed through co-expression of specific affinity molecules designed for entrapment in the endoplasmic reticulum during the export process. We describe the investigation of a small (6 kDa) non-immunoglobulin-based HER2 receptor binding affibody molecule (ZHER2:00477), for use in affinity mediated entrapment of the HER2 receptor in the ER. Constructs encoding ZHER2:00477 or a control affibody protein, with or without ER-retention peptide extensions (KDEL), were expressed in the HER2 over-expressing cell line SKOV-3. Intracellular expression of the full-length affibody constructs could be confirmed by probing cell extracts by Western blotting. Confocal immunofluorescence microscopy experiments showed extensive co-localization of the HER2 receptor and ZHER2:00477-KDEL in the ER, whereas the use of a KDEL-extended control affibody molecule resulted in distinct and separate signals from cell surface-localized HER2 receptor and ER-localized affibody protein. This indicated a capability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2 receptor in a specific manner. Using flow cytometry and cell proliferation analyses, it could be shown that expression of the ZHER2:00477-KDEL fusion construct in the SKOV-3 cell line resulted both in a marked reduction in cell surface level of HER2 receptors and that the cell population doubling time was significantly increased. Expression of the ZHER2:00477-KDEL fusion protein in additional cell lines of different origin and with different expression levels of endogenous HER2 receptor compared to SKOV-3, also resulted in depletion of the cell surface levels of HER2 receptor. This indicated upon a general ability of the ZHER2:00477-KDEL fusion protein to functionally interfere with the export process of HER2.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

Nyckelord

ErbB2; HER2; affibody molecule; endoplasmic reticulum; flow cytometry

Bioengineering

Bioteknik

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