High-Throughput Protein Production of Membrane Proteins in Saccharomyces cerevisiae.

This chapter outlines a protocol to assess viability for large-scale protein production and purification for selected targets from an initial medium-throughput cloning strategy. Thus, one can assess a broad number of potential candidate proteins, mutants, or expression variants using an empirically minimalistic approach. In addition, a key output from this protocol is utilization of Saccharomyces cerevisiae as a means for the efficient screening and production of purified proteins. The primary focus in this protocol is overexpression of polytopic integral membrane proteins though methods can be equally applied to soluble proteins. The protocol starts with outlining high-throughput (sans robotics) cloning of expression proteins into a dual-tag yeast expression plasmid. These membrane proteins are then screened for expression level, detergent solubilization, initial purity, and chromatography characteristics. Both small- and large-scale expression methods are discussed along with fermentation.