mRNA display selection of a high-affinity, modification-specific phospho-IkappaBalpha-binding fibronectin.
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 IkappaBalpha. 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-IkappaBalpha peptide with K d = 18 nM and is over 1000-fold specific compared to the nonphosphorylated peptide. 10C17C25 specifically recognizes endogenous phosphorylated IkappaBalpha from mammalian cell extract and stabilizes phospho-IkappaBalpha in vivo. We also incorporated 10C17C25 into a FRET indicator that detects IkappaB kinase (IKK) activity in vitro, demonstrating the utility of selecting designed adaptors for kinase activity sensors.
Olson
C Anders
CA
Biochemistry and Molecular Biophysics Option, California Institute of Technology, Pasadena, California 91125, USA.
Liao
Hsiang-I
HI
Sun
Ren
R
Roberts
Richard W
RW
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D019151:Peptide Library; D010766:Phosphorylation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D020543:Proteome / Q000096:biosynthesis / Q000235:genetics; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance
D005353:Fibronectins; D008024:Ligands; D010446:Peptide Fragments; D019151:Peptide Library; D020543:Proteome; D012333:RNA, Messenger; D051550:I-kappa B Kinase