Author(s): Deshpande A, Putcha BD, Kuruganti S, Walter MR
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Abstract 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 KD 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. © 2013 The Protein Society.
This article was published in Protein Sci
and referenced in Journal of Clinical & Cellular Immunology