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- W1586315430 abstract "We have developed a new approach based on the Gγ recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Gγ 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 γ subunit (Gγ) is essential for signal transduction in yeast. In the original Y2H system, an engineered Gγ that lacks membrane localization upon deletion of the lipid modification site (Gγcyto) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Gγcyto are expressed. As protein–protein interactions bring Gγcyto 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–Gγcyto 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 Gγ recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones. Structured digital abstract • MINT-7556266: Fc portion of human IgG (uniprotkb:P01857) physically interacts (MI:0915) with Z domain of protein A (uniprotkb:P38507) by two hybrid (MI:0018)" @default.
- W1586315430 created "2016-06-24" @default.
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- W1586315430 date "2010-03-01" @default.
- W1586315430 modified "2023-10-18" @default.
- W1586315430 title "The competitor-introduced Gγ recruitment system, a new approach for screening affinity-enhanced proteins" @default.
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- W1586315430 doi "https://doi.org/10.1111/j.1742-4658.2010.07592.x" @default.
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