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

Fingerprint

Tumor Biomarkers
Visualization
Surface Plasmon Resonance
Proteins
Surface plasmon resonance
ErbB-2 Receptor
Immunoglobulin Fragments
Immunoglobulin Isotypes
Affinity chromatography
Protein Transport
Fluorescent Dyes
Affinity Chromatography
Epidermal Growth Factor Receptor
Confocal Microscopy
Confocal microscopy
red fluorescent protein
Assays
Agglomeration

Keywords

  • DARPin
  • mCherry
  • tumor marker HER2/neu

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)

Cite this

Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu. / Mironova, K. E.; Chernykh, O. N.; Ryabova, A. V.; Stremovskiy, O. A.; Proshkina, G. M.; Deyev, S. M.

In: Biochemistry (Moscow), Vol. 79, No. 12, 13.12.2014, p. 1391-1396.

Research output: Contribution to journalArticle

Mironova, K. E. ; Chernykh, O. N. ; Ryabova, A. V. ; Stremovskiy, O. A. ; Proshkina, G. M. ; Deyev, S. M. / Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu. In: Biochemistry (Moscow). 2014 ; Vol. 79, No. 12. pp. 1391-1396.
@article{27c203b5750d40fe9bde1bad915a3bbe,
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.",
keywords = "DARPin, mCherry, tumor marker HER2/neu",
author = "Mironova, {K. E.} and Chernykh, {O. N.} and Ryabova, {A. V.} and Stremovskiy, {O. A.} and Proshkina, {G. M.} and Deyev, {S. M.}",
year = "2014",
month = "12",
day = "13",
doi = "10.1134/S0006297914120141",
language = "English",
volume = "79",
pages = "1391--1396",
journal = "Biochemistry. Biokhimiia",
issn = "0006-2979",
publisher = "Maik Nauka-Interperiodica Publishing",
number = "12",

}

TY - JOUR

T1 - Highly specific hybrid protein DARPin-mCherry for fluorescent visualization of cells overexpressing tumor marker HER2/neu

AU - Mironova, K. E.

AU - Chernykh, O. N.

AU - Ryabova, A. V.

AU - Stremovskiy, O. A.

AU - Proshkina, G. M.

AU - Deyev, S. M.

PY - 2014/12/13

Y1 - 2014/12/13

N2 - 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.

AB - 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.

KW - DARPin

KW - mCherry

KW - tumor marker HER2/neu

UR - http://www.scopus.com/inward/record.url?scp=84918550793&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84918550793&partnerID=8YFLogxK

U2 - 10.1134/S0006297914120141

DO - 10.1134/S0006297914120141

M3 - Article

VL - 79

SP - 1391

EP - 1396

JO - Biochemistry. Biokhimiia

JF - Biochemistry. Biokhimiia

SN - 0006-2979

IS - 12

ER -