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