Membrane protein expression and analysis in yeast.

This protocol describes the expression and analysis of membrane proteins produced in yeast, as illustrated with Yarrowia lipolytica and Pichia pastoris. Step by step, we explain how to generate a yeast strain expressing the membrane protein of interest, how to prepare a membrane protein sample from yeast, and how to analyze the expression levels using SDS-PAGE and Western blotting. In the final section, we describe how to perform a radioligand binding assay, which quantifies the amount of the protein folded in a ligand-binding competent state.

Enhanced membrane protein expression by engineering increased intracellular membrane production.

BACKGROUND: Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. RESULTS: We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. CONCLUSIONS: We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.

The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins.

BACKGROUND: The unfolded protein response (UPR) in eukaryotes upregulates factors that restore ER homeostasis upon protein folding stress and in yeast is activated by a non-conventional splicing of the HAC1 mRNA. The spliced HAC1 mRNA encodes an active transcription factor that binds to UPR-responsive elements in the promoter of UPR target genes. Overexpression of the HAC1 gene of S. cerevisiae can reportedly lead to increased production of heterologous proteins. To further such studies in the biotechnology favored yeast Pichia pastoris, we cloned and characterized the P. pastoris HAC1 gene and the splice event. RESULTS: We identified the HAC1 homologue of P. pastoris and its splice sites. Surprisingly, we could not find evidence for the non-spliced HAC1 mRNA when P. pastoris was cultivated in a standard growth medium without any endoplasmic reticulum stress inducers, indicating that the UPR is constitutively active to some extent in this organism. After identification of the sequence encoding active Hac1p we evaluated the effect of its overexpression in Pichia. The KAR2 UPR-responsive gene was strongly upregulated. Electron microscopy revealed an expansion of the intracellular membranes in Hac1p-overexpressing strains. We then evaluated the effect of inducible and constitutive UPR induction on the production of secreted, surface displayed and membrane proteins. Wherever Hac1p overexpression affected heterologous protein expression levels, this effect was always stronger when Hac1p expression was inducible rather than constitutive. Depending on the heterologous protein, co-expression of Hac1p increased, decreased or had no effect on expression level. Moreover, alpha-mating factor prepro signal processing of a G-protein coupled receptor was more efficient with Hac1p overexpression; resulting in a significantly improved homogeneity. CONCLUSIONS: Overexpression of P. pastoris Hac1p can be used to increase the production of heterologous proteins but needs to be evaluated on a case by case basis. Inducible Hac1p expression is more effective than constitutive expression. Correct processing and thus homogeneity of proteins that are difficult to express, such as GPCRs, can be increased by co-expression with Hac1p.