Isolation of Single-Domain Antibodies to Transmembrane Proteins Using Magnetized Yeast Cell Targets.

The isolation of binding ligands from yeast-displayed combinatorial libraries has typically relied on the use of a soluble, recombinantly expressed form of the target protein when performing magnetic selections or fluorescence-activated cell sorting. When identifying binding ligands, appropriate target protein expression and subsequent purification represents a significant bottleneck. As an alternative, we describe the use of target proteins expressed on the surface of magnetized yeast cells in the selection of yeast-displayed nanobody libraries. In this approach, yeast cells displaying the target protein also co-express an iron oxide-binding protein; incubation with iron oxide nanopowder results in magnetization of target-displaying cells. Alternatively, target-displaying cells are magnetized by nonspecific adsorption of iron oxide nanopowder. Subsequently, any library cells that interact with the magnetized target cells can be isolated using a magnet. Here, we detail protocols for the isolation of binders to membrane protein targets from a yeast display nanobody library using magnetized yeast cell targets. We provide guidance on how to generate magnetic yeast cell targets as well as library selection conditions to bias the isolation of high affinity binders. We also discuss how to assess the affinity and specificity of the isolated nanobodies using flow cytometry.