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Published August 15, 2008 | Supplemental Material + Accepted Version
Journal Article Open

mRNA Display Selection of a High-Affinity, Modification-Specific Phospho-IκBα-Binding Fibronectin

Abstract

The complexity of the human proteome is greatly expanded by post-translational modifications. New tools capable of recognizing these modifications in a sequence-specific fashion provide a route to purify these modified proteins, to alter protein trafficking, and to visualize signal transduction in real time. Here, we have evolved novel, modification-specific ligands that target phosphorylated IκBα. To do this, we employed mRNA display-based in vitro selection using a 30-trillion-member protein library based on the fibronectin type III domain. The selection yielded one fibronectin molecule, 10C17C25, that binds a phospho-IκBα peptide with Kd = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IκBα from mammalian cell extract and stabilizes phospho-IκBα in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IκB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.

Additional Information

© 2008 American Chemical Society. Received 24 March 2008. Date accepted 2 June 2008. Published online 1 July 2008. Published in print 1 August 2008. We thank Dr. T. Leung and Dr. M. Boldin (Caltech) for advice. We thank Dr. Geoffrey S. Waldo (Los Alamos National Laboratory) for donation of the GFP reporter vector and Dr. Jose Aberola-Ila (Oklahoma Medical Research Foundation) for donation of ECFP and EYFP plasmids. C.A.O. was supported by an NSF graduate fellowship. Funding was provided by NIH RO1 GM60416 (R.W.R.), and the American Foundation for AIDS Research (R.W.R.)

Attached Files

Accepted Version - nihms127064.pdf

Supplemental Material - cb800069c-file006.pdf

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Additional details

Created:
August 19, 2023
Modified:
October 24, 2023