Isolating and Engineering Fluorescence-Activating Proteins Using Yeast Surface Display

Abstract

This protocol describes the workflow to isolate and engineer fluorescence-activating proteins by yeast surface display. Fluorescence-activating proteins are an emerging class of fluorescent chemogenetic reporters for monitoring gene expression and protein localization in living cells and organisms. They become fluorescent upon binding exogenously applied fluorogenic organic dyes. Efficient fluorescence-activating proteins can be selected from yeast-displayed libraries by iterative rounds of fluorescence-activated cell sorting. The overall strategy is described, as well as a strategy for characterizing the affinity and spectroscopic properties of the selected clones.

A tunable and versatile chemogenetic near infrared fluorescent reporter

“…To increase molecular brightness and fluorogen binding affinity, we used yeast surface display 8 to evolve a protein variant able to efficiently bind and stabilize only the deprotonated state of HPAR-3,5DOM (see also Supplementary Text 1 ). We created a combinatorial library of variants, which we expressed at the surface of yeast cells.…”