The synthesis of recombinant membrane proteins in yeast for structural studies

Historically, recombinant membrane protein production has been a major challenge meaning that many fewer membrane protein structures have been published than those of soluble proteins. However, there has been a recent, almost exponential increase in the number of membrane protein structures being deposited in the Protein Data Bank. This suggests that empirical methods are now available that can ensure the required protein supply for these difficult targets. This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins. We provide an overview of approaches to optimize the expression plasmid, host cell and culture conditions, as well as the extraction and purification of functional protein for crystallization trials in preparation for structural studies.

Membrane protein production in the Yeast, S. cerevisiae

The first crystal structures of recombinant mammalian membrane proteins were solved in 2005 using protein that had been produced in yeast cells. One of these, the rabbit Ca2+-ATPase SERCA1a, was synthesized in Saccharomyces cerevisiae. All host systems have their specific advantages and disadvantages, but yeast has remained a consistently popular choice in the eukaryotic membrane protein field because it is quick, easy and cheap to culture, whilst being able to post-translationally process eukaryotic membrane proteins. Very recent structures of recombinant membrane proteins produced in S. cerevisiae include those of the Arabidopsis thaliana NRT1.1 nitrate transporter and the fungal plant pathogen lipid scramblase, TMEM16. This chapter provides an overview of the methodological approaches underpinning these successes.