A modular design of low-background bioassays based on a high-affinity molecular pair barstar: Barnase

Varun K A Sreenivasan, Timothy A. Kelf, Ekaterina A. Grebenik, Oleg A. Stremovskiy, Jana M. Say, James R. Rabeau, Andrei V. Zvyagin, Sergey M. Deyev

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

High-affinity molecular pairs provide a convenient and flexible modular base for the design of molecular probes and protein/antigen assays. Specificity and sensitivity performance indicators of a bioassay critically depend on the dissociation constant (KD) of the molecular pair, with avidin:biotin being the state-of-the-art molecular pair (KD ~ 1 fM) used almost universally for applications in the fields of nanotechnology and proteomics. In this paper, we present an alternative high-affinity protein pair, barstar:barnase (KD ~ 10 fM), which addresses several shortfalls of the avidin:biotin system, including non-negligible background due to the non-specific binding. A quantitative assessment of the non-specific binding carried out using a model assay revealed inherent irreproducibility of the [strept]avidin:biotin-based assays, attributed to the avidin binding to solid phases, endogenous biotin molecules and serum proteins. On the other hand, the model assays assembled via a barstar:barnase protein linker proved to be immune to such non-specific binding, showing good prospects for high-sensitivity rare biomolecular event nanoproteomic assays.

Original languageEnglish
Pages (from-to)1437-1443
Number of pages7
JournalProteomics
Volume13
Issue number9
DOIs
Publication statusPublished - May 2013
Externally publishedYes

Keywords

  • Avidin
  • Barnase
  • Barstar
  • Biotin
  • Nanoproteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Sreenivasan, V. K. A., Kelf, T. A., Grebenik, E. A., Stremovskiy, O. A., Say, J. M., Rabeau, J. R., Zvyagin, A. V., & Deyev, S. M. (2013). A modular design of low-background bioassays based on a high-affinity molecular pair barstar: Barnase. Proteomics, 13(9), 1437-1443. https://doi.org/10.1002/pmic.201200491