title: Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein-protein interactions.
abstract: Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gγ recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gγ recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 × 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wild-type haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification.
authors: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada-ku, Kobe, Japan.
authors: Nobuo
authors: N
authors: Fukuda
authors: Jun
authors: J
authors: Ishii
authors: Akihiko
authors: A
authors: Kondo
mesh_terms: D019943:Amino Acid Substitution; D015169:Colony Count, Microbial; D004171:Diploidy; D044388:GTP-Binding Protein gamma Subunits / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012100:Reproduction, Asexual; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism
chemical_list: D044388:GTP-Binding Protein gamma Subunits; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D050505:Mutant Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C084294:STE18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins