title: 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.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Misato
authors.initials: M
authors.lastname: Kaishima
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Fukuda
authors.affiliation: Organization of Advanced Science and Technology, Kobe University, Kobe, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D029701:Saccharomyces cerevisiae Proteins