Desired Alteration of Protein Affinities: Competitive Selection of Protein Variants Using Yeast Signal Transduction Machinery

Abstract

Molecules that can control protein-protein interactions (PPIs) have recently drawn attention as new drug pipeline compounds. Here, we report a technique to screen desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. We previously constructed a screening system based on a target protein fused to a mutated G-protein γ subunit (Gγcyto) lacking membrane localization ability. This ability, required for signal transmission, is restored by recruiting Gγcyto into the membrane only when the target protein interacts with an artificially membrane-anchored candidate protein, thereby allowing interacting partners (Gγ recruitment system) to be searched and identified. In the present study, the Gγ recruitment system was altered by integrating the cytosolic expression of a third protein as a competitor to set a desirable affinity threshold. This enabled the reliable selection of both affinity-enhanced and affinity-attenuated protein variants. The presented approach may facilitate the development of therapeutic proteins that allow the control of PPIs.

Expression of varied GFPs in Saccharomyces cerevisiae: codon optimization yields stronger than expected expression and fluorescence intensity

“…FIG1 gene expression is induced in response to activation of the yeast mating signaling pathway; therefore, this gene has been often used to sense the signal promoted in the mating pathway (Fig. 2A) or the agonist response to heterologous GPCRs using the non-codon-optimized EGFP as the reporter gene38394041.…”

“…We developed the Gγ recruitment system to detect protein–protein interactions (PPIs)4042434445 and to screen for protein variants presenting desirable affinities40. The detection method of the Gγ recruitment system is based on the fundamental principle that yeast pheromone (mating) signaling requires the localization of a complex between guanine nucleotide binding protein (G-protein) β- and γ-subunits (Gβγ) and the inner leaflet of the plasma membrane46.…”

“…When the target protein (X) is a soluble cytosolic protein, Gγ cyto is prepared as a fusion protein with the target protein (X) (Gγ cyto -X), and library proteins (Y) are attached to the artificial lipidation site to localize the proteins to the membrane (Supplementary Fig. S3A)4042. In contrast, when the target protein (X) is a membrane-bound protein, Gγ cyto is prepared as a fused protein with the library proteins (Y) (Gγ cyto -Y) (Supplementary Fig.…”

“…2A) were conducted according to previously described methods40 with some modifications. The engineered yeast a -cells (Supplementary Table S3) were grown in 5 mL of YPD medium 30 °C overnight.…”

“…Protein–protein interaction assays using FIG1-GFP reporter genes were conducted as described previously40. In brief, for the Gγ recruitment system involving soluble cytosolic target protein (Fig.…”