title: The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.
abstract: We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Fukuda
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Tanaka
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004252:DNA Mutational Analysis; D005434:Flow Cytometry / Q000379:methods; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008055:Lipids / Q000737:chemistry; D008957:Models, Genetic; D009154:Mutation; D025941:Protein Interaction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D015398:Signal Transduction; D013211:Staphylococcus aureus / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D044388:GTP-Binding Protein gamma Subunits; D008055:Lipids; D049452:Green Fluorescent Proteins