Overproduction of Membrane-Associated, and Integrated, Proteins Using Saccharomyces cerevisiae.

Abstract

Structural and functional eukaryotic membrane protein research continues to grow at an increasing rate, placing greater significance on leveraging productive protein expression pipelines to feed downstream studies. Bacterial expression systems (e.g., E. coli) are often the preferred system due to their simple growth conditions, relative simplicity in experimental workflow, low overall cost per liter of cell growth, and ease of genetic manipulation. However, overproduction success of eukaryotic membrane proteins in bacterial systems is hindered by the limited native processing ability of bacterial systems for important protein folding interactions (e.g., disulfide bonds), post-translational modifications (e.g., glycosylation), and inherent disadvantages in protein trafficking and folding machinery compared to other expression systems.In contrast, Saccharomyces cerevisiae expression systems combine positive benefits of simpler bacterial systems with those of more complex eukaryotic systems (e.g., mammalian cells). Benefits include inexpensive growth, robust DNA repair and recombination machinery, amenability to high density growths in bioreactors, efficient transformation, and robust post-translational modification machinery. These characteristics make S. cerevisiae a viable first-alternative when bacterial overproduction is insufficient. Thus, this chapter provides a framework, using methods that have proven successful in prior efforts, for overproducing membrane anchored or membrane integrated proteins in S. cerevisiae. The framework is designed to improve yields for all levels of overexpression expertise, providing optimization insights for the variety of processes involved in heterologous protein expression.