A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities.

ABSTRACT: We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 μm After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical

SUBMITTER: Cluet D 

PROVIDER: S-EPMC7124468 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature