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Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein‐protein interactions
Author(s) -
Fukuda Nobuo,
Ishii Jun,
Kondo Akihiko
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08232.x
Subject(s) - mutant , transient (computer programming) , biology , mating of yeast , signal transduction , signal (programming language) , saccharomyces cerevisiae , microbiology and biotechnology , yeast , protein–protein interaction , genetics , computational biology , gene , computer science , programming language , operating system
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.