title: Heterologous expression of G-protein-coupled receptors in yeast.
abstract: Heterologous yeast expression systems have been successfully used for the production of G-protein-coupled receptors (GPCRs) for both structural and functional studies. Yeast combine comparatively low cost and short culture times with straightforward generation of expression clones. They also perform some key posttranslational modifications not possible in bacterial systems. There are two major yeast expression systems, Pichia pastoris and Saccharomyces cerevisiae, both of which have been used for the production of GPCRs. P. pastoris has a proven track record for the production of large amounts of GPCR for structural studies. High-resolution crystal structures of both the adenosine A2A and the histamine H1 receptors have been obtained using protein expressed in this system. S. cerevisiae is relatively easy to engineer and this has resulted in the development of sophisticated tools for the functional characterization of GPCRs. In this chapter, we provide protocols for both large-scale receptor expression in P. pastoris for structural studies and small-scale receptor expression in S. cerevisiae for functional characterization. In both cases, the receptor used is the human adenosine A2A receptor. The results that both we and others have obtained using these protocols show the wide utility of the yeast expression systems for the production of GPCRs.
authors: Department of Life Sciences, Imperial College London, London, United Kingdom.
authors: Nicolas
authors: N
authors: Bertheleme
authors: Department of Life Sciences, Imperial College London, London, United Kingdom.
authors: Shweta
authors: S
authors: Singh
authors: Department of Molecular Discovery Research, GlaxoSmithKline, Hertfordshire, United Kingdom.
authors: Simon
authors: S
authors: Dowell
authors: Department of Life Sciences, Imperial College London, London, United Kingdom. Electronic address: b.byrne@imperial.ac.uk.
authors: Bernadette
authors: B
authors: Byrne
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods; D010843:Pichia / Q000235:genetics* / Q000254:growth & development; D010957:Plasmids / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000032:analysis / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
chemical_list: D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins
keywords: G-protein signaling; G-protein-coupled receptor; Heterologous protein expression; Pichia pastoris; Radioligand binding analysis; Saccharomyces cerevisiae; Yeast