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A modular design of low‐background bioassays based on a high‐affinity molecular pair barstar:barnase
Author(s) -
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.
Publication year - 2013
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201200491
Subject(s) - avidin , barnase , biotin , molecular binding , dissociation constant , chemistry , computational biology , protein engineering , immunoassay , biochemistry , linker , streptavidin , biophysics , biology , molecule , antibody , ribonuclease , computer science , enzyme , genetics , rna , receptor , organic chemistry , gene , operating system
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 ( K D ) of the molecular pair, with avidin:biotin being the state‐of‐the‐art molecular pair ( K D ∼ 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 ( K D ∼ 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.