Disulfide Trapping of Protein Complexes on the Yeast Surface

Protein complexes are common in nature but studying protein association in vitro is difficult because it often involves lengthy and expensive biochemical steps. There are also technical difficulties obtaining the required sensitivity and resolution of the assays. Here, we introduce a combination of yeast display and disulfide trapping that can be used to detect the formation of a broad spectrum of protein complexes on the yeast surface using flow cytometry without protein purification. The technique is based on co-expressing two potentially interacting proteins in the same yeast cell, one of which is designed to be anchored to the cell wall and the other is designed to be in a soluble form. Importantly, we introduce a structure-based disulfide between the subunits to covalently crosslink them so that the bound complex can be efficiently detected irrespective of the stability of the complex. Without such crosslinking, most protein complexes dissociate before they can be detected on the cell surface. We show that the technique is general by applying it to trap a diverse group of complexes, including both folded subunits and disordered peptides with an affinity ranging from 100 nM – 20 mM. The method can be used to assist in the testing of rationally designed interactions as well as to develop a directed evolution study to find novel protein inhibitors.