Highly Specific Hybrid Protein DARPin-mCherry for Fluorescent Visualization of Cells Overexpressing Tumor Marker HER2/neu

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K. E. Mironova^1,2*, O. N. Chernykh^1,3, A. V. Ryabova⁴, O. A. Stremovskiy¹, G. M. Proshkina¹, and S. M. Deyev^1,2

¹Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: +7 (495) 335-0812; E-mail: office@ibch.ru; kgobova@gmail.com

²Lobachevsky State University of Nizhni Novgorod, pr. Gagarina 23, 603950 Nizhni Novgorod, Russia

³Lomonosov Moscow State University, Faculty of Biology, 119234 Moscow, Russia

⁴Prokhorov General Physics Institute, Russian Academy of Sciences, ul. Vavilova 38, 119991 Moscow, Russia; fax: +7 (499) 135-0270

^* To whom correspondence should be addressed.

Received July 20, 2014; Revision received September 3, 2014

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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.

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KEY WORDS: DARPin, mCherry, tumor marker HER2/neu

DOI: 10.1134/S0006297914120141

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