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

Fingerprint

Avidin
Bioassay
Biotin
Biological Assay
Assays
Molecular Probes
Proteins
Nanotechnology
Proteomics
Blood Proteins
Antigens
Sensitivity and Specificity
Bacillus amyloliquefaciens barstar protein
Bacillus amyloliquefaciens ribonuclease
Molecules

Keywords

  • Avidin
  • Barnase
  • Barstar
  • Biotin
  • Nanoproteomics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Sreenivasan, V. K. A., Kelf, T. A., Grebenik, E. A., Stremovskiy, O. A., Say, J. M., Rabeau, J. R., ... 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

A modular design of low-background bioassays based on a high-affinity molecular pair barstar : Barnase. / Sreenivasan, Varun K A; Kelf, Timothy A.; Grebenik, Ekaterina A.; Stremovskiy, Oleg A.; Say, Jana M.; Rabeau, James R.; Zvyagin, Andrei V.; Deyev, Sergey M.

In: Proteomics, Vol. 13, No. 9, 05.2013, p. 1437-1443.

Research output: Contribution to journalArticle

Sreenivasan, VKA, Kelf, TA, Grebenik, EA, Stremovskiy, OA, Say, JM, Rabeau, JR, Zvyagin, AV & Deyev, SM 2013, 'A modular design of low-background bioassays based on a high-affinity molecular pair barstar: Barnase', Proteomics, vol. 13, no. 9, pp. 1437-1443. https://doi.org/10.1002/pmic.201200491
Sreenivasan VKA, Kelf TA, Grebenik EA, Stremovskiy OA, Say JM, Rabeau JR et al. A modular design of low-background bioassays based on a high-affinity molecular pair barstar: Barnase. Proteomics. 2013 May;13(9):1437-1443. https://doi.org/10.1002/pmic.201200491
Sreenivasan, Varun K A ; Kelf, Timothy A. ; Grebenik, Ekaterina A. ; Stremovskiy, Oleg A. ; Say, Jana M. ; Rabeau, James R. ; Zvyagin, Andrei V. ; Deyev, Sergey M. / A modular design of low-background bioassays based on a high-affinity molecular pair barstar : Barnase. In: Proteomics. 2013 ; Vol. 13, No. 9. pp. 1437-1443.
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