Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu

K. E. Mironova, O. N. Chernykh, A. V. Ryabova, O. A. Stremovskiy, G. M. Proshkina, S. M. Deyev

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Here we propose a simple and reliable approach for detection of the tumor marker HER2/neu using the targeting fluorescent hybrid protein DARPin-mCherry. As a targeting module, we used DARPin9-29, which is a member of a novel class of non-immunoglobulin targeting proteins that can highly selectively recognize the extracellular domain of the epidermal growth factor receptor HER2/neu. The red fluorescent protein mCherry was used as the detecting module. The hybrid protein DARPin-mCherry was prepared with high yield in a bacterial expression system and purified in one step by affinity chromatography. The purified protein is not prone to aggregation. The specificity of DARPin-mCherry binding with the HER2/neu tumor marker was demonstrated using confocal microscopy, flow cytofluorimetry, and surface plasmon resonance. The dissociation constant of the DARPin-mCherry protein complex with the HER2/neu receptor determined by surface plasmon resonance was calculated to be 4.5 nM. These characteristics of the hybrid protein DARPin-mCherry suggest it as a promising agent for immunofluorescent assay and an attractive alternative to antibodies and their fragments labeled with fluorescent dyes that are now used for this purpose.

Original languageEnglish
Pages (from-to)1391-1396
Number of pages6
JournalBiochemistry (Moscow)
Volume79
Issue number12
DOIs
Publication statusPublished - 13 Dec 2014
Externally publishedYes

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Keywords

  • DARPin
  • mCherry
  • tumor marker HER2/neu

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

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