title: Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu.
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.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia. office@ibch.ru.
authors.forename: K E
authors.initials: KE
authors.lastname: Mironova
authors.forename: O N
authors.initials: ON
authors.lastname: Chernykh
authors.forename: A V
authors.initials: AV
authors.lastname: Ryabova
authors.forename: O A
authors.initials: OA
authors.lastname: Stremovskiy
authors.forename: G M
authors.initials: GM
authors.lastname: Proshkina
authors.forename: S M
authors.initials: SM
authors.lastname: Deyev
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D057054:Molecular Imaging / Q000379:methods*; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D000097573:Red Fluorescent Protein
chemical_list: D014408:Biomarkers, Tumor; D008164:Luminescent Proteins; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein