Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins

Abstract

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae . We demonstrate that our S. cerevisiae -based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli . We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.

The structural basis of divalent cation block in a tetrameric prokaryotic sodium channel

“…6a and Table 2 ). These current values are consistent with the previously reported single channel recording experimental data of NavAb or BacNavs 31 , 32 . Therefore, the simulation applying the ECC well represented the actual situation.…”

Production of an Active, Human Membrane Protein in Saccharomyces cerevisiae: Full-Length FICD

“…Obtaining sufficient quantities of pure membrane protein can be both challenging and time consuming due to low expression levels, solubility issues and challenges in obtaining and maintaining uniformly folded protein. To overcome some of these challenges, we employed the previously developed PAP1500 S. cerevisiae -based membrane protein expression platform [ 31 , 32 , 33 , 34 , 35 , 36 ], and coupled this with GFP tagging to produce full-length human FICD ( Figure 2 ). This platform has been successfully used to produce multi-spanning membrane proteins such as aquaporins, transmembrane ATPases and ion channels [ 32 , 33 , 34 , 35 , 37 , 38 , 39 , 40 ].…”