Heterologous expression of human membrane receptors in the yeast Saccharomyces cerevisiae.
Due to their implication in numerous diseases like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension, and Alzheimer disease, membrane proteins (MPs) represent around 50% of drug targets. However, only 204 crystal structures of MPs have been solved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. The yeast Saccharomyces cerevisiae is a convenient host for the production of mammalian MPs for functional and structural studies. Like bacteria, they are straightforward to manipulate genetically, are well characterized, can be easily cultured, and can be grown inexpensively in large quantities. The advantage of yeast compared to bacteria is that they have protein-processing and posttranslational modification mechanisms related to those found in mammalian cells. The recombinant rabbit muscle Ca(2+)-ATPase (adenosine triphosphatase), the first heterologously expressed mammalian MP for which the crystal structure was resolved, has been produced in S. cerevisiae. In this chapter, the focus is on expression of recombinant human integral MPs in a functional state at the plasma membrane of the yeast S. cerevisiae. Optimization of yeast culture and of MP preparations is detailed for two human receptors of the Hedgehog pathway: Patched and Smoothened.
Joubert
Olivier
O
Université Henri Poincaré- Nancy 1, France.
Nehmé
Rony
R
Bidet
Michel
M
Mus-Veteau
Isabelle
I
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000072079:Patched Receptors; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D000072086:Smoothened Receptor
D008565:Membrane Proteins; D000072079:Patched Receptors; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C491023:SMO protein, human; D000072086:Smoothened Receptor