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Kinetic analysis of cytokine‐mediated receptor assembly using engineered FC heterodimers
Author(s) -
Deshpande Ashlesha,
Putcha BalanandaDhurjati Kumar,
Kuruganti Srilalitha,
Walter Mark R.
Publication year - 2013
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2285
Subject(s) - surface plasmon resonance , receptor , receptor–ligand kinetics , ligand (biochemistry) , biophysics , chemistry , kinetics , covalent bond , fusion protein , biology , biochemistry , recombinant dna , nanotechnology , materials science , physics , organic chemistry , quantum mechanics , nanoparticle , gene
A method for analyzing ligand–receptor binding kinetics is described, which is based on an engineered FC domain (FChk) that forms a covalent heterodimer. To validate the system, the type I IFN receptors (IFNAR1 and IFNAR2) were expressed as IFNAR1‐FChk, IFNAR2‐FCkh, and IFNAR1/IFNAR2‐FChk fusion proteins. Surface plasmon resonance (SPR) analysis of binary IFNα2a/IFNAR interactions confirmed prior affinity measurements, while the affinity of the IFNα2a/IFNAR1/IFNAR2‐FChk interaction reproduced the affinity of IFNα2a binding to living cells. In cellular assays, IFNAR1/IFNAR2‐FChk potently neutralized IFNα2a bioactivity with an inhibitory concentration equivalent to the K D measured by SPR. These studies suggest that FChk provides a simple reagent to evaluate the binding kinetics of multiple ligand–receptor signaling systems that control cell growth, development, and immunity.