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Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands
journal contributionposted on 2020-10-22, 15:33 authored by Kaitlyn Bacon, John Bowen, Hannah Reese, Balaji M. Rao, Stefano Menegatti
This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of select WW-YAP-binding peptides: KD values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.
WW4 μ MYes-Associated Protein 1target-binding mRNA-peptide fusionsScreening mRNA-Display Peptide Libr...3 μ MWW-YAPEquilibrium adsorption studiesTOM 22iron oxide nanoparticlesTarget-Displaying Magnetized Yeastscreening mRNA-display librariesmitochondrial import receptor subun...yeast-displayed protein targetsK D valuescyclo