Abstract
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.
Highlights
The synthesis of recombinant membrane proteins in yeast for structural studies Sarah J
This review focuses on methods that are available for protein production in yeast, which is an important source of recombinant eukaryotic membrane proteins
The first crystal structures of mammalian membrane proteins derived from recombinant sources were solved in 2005 using protein that had been produced in yeast cells: the rabbit Ca2+-ATPase, SERCA1a, was overexpressed in Saccharomyces cerevisiae [1] and the rat voltage-dependent potassium ion channel, Kv1.2 was produced in Pichia pastoris [2] (Fig. 1a and e)
Summary
The first crystal structures of mammalian membrane proteins derived from recombinant sources were solved in 2005 using protein that had been produced in yeast cells: the rabbit Ca2+-ATPase, SERCA1a, was overexpressed in Saccharomyces cerevisiae [1] and the rat voltage-dependent potassium ion channel, Kv1.2 was produced in Pichia pastoris [2] (Fig. 1a and e). Structures of the Arabidopsis thaliana NRT1.1 nitrate transporter, a fungal plant pathogen TMEM16 lipid scramblase and the yeast mitochondrial ADP/ATP carrier were solved using recombinant protein produced in S. cerevisiae (Fig. 1b–d, f–k). Despite these successes (as well as others using recombinant protein produced in bacteria, insect cells and mammalian celllines; see http://blanco.biomol.uci.edu/mpstruc/), the overall rate of progress in membrane protein structural biology has, until very recently, been markedly slower than that in the soluble protein field [6]. This review focuses on current approaches to optimizing expression plasmids, yeast strains and culture conditions, as well as the extraction and purification of functional membrane proteins for crystallization trials (and subsequent structural studies) using detergents and SMA co-polymers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
