Enzymatic Modification and Flow Cytometry Assessment of Yeast Surface Displayed Proteins

Yeast surface display is a genotype-phenotype linkage strategy that empowers high-throughput screening of protein function. Traditionally, yeast surface display has been applied to the evolution of new binding proteins, with flow cytometry used to assess and sort by levels of binding strength. Recently, there has been growing interest in applying yeast surface display for screening enzymatic modification of substrate variants, with additive (e.g., phosphorylation) or subtractive (e.g., proteolysis) modifications providing a phenotype readable by flow cytometry. Such modifications are regularly applied using intracellular co-localization, but the ability to achieve extracellular enzymatic modification of displayed substrates could open many more reactions to investigation. Here, we describe techniques for designing and applying screening assays for extracellular enzymatic modification to candidate substrates displayed on the yeast surface and subsequent evaluation using flow cytometry analysis. We provide these protocols in the context of phosphatases dephosphorylating yeast displayed substrates containing phosphorylated tyrosine residues and comment on how this applied framework can be adapted to developing screening assays for other enzyme-substrate pairs.