Production of adrenergic receptors in yeast.
Using a recombinant yeast strain expressing human beta 2 adrenergic receptors under a galactose-inducible promoter, we established conditions for receptor production in 1-15 liter fermenter culture. Crucial factors contributing to consistent high-level expression included the use of selective glucose-free medium, the maintenance of the pH of the culture at 7.2-7.5 and the presence of an antagonist. The expression strategy and production conditions used with the beta 2 adrenergic receptor were then employed to express the human alpha 2-C2 adrenergic receptor in Saccharomyces cerevisiae. Galactose-induced yeast cells displayed specific, high-affinity [3H]rauwolscine binding and contained a 50-kDa species recognized by an alpha 2-C2 receptor specific antiserum. In fermenter culture, up to 10(5) high-affinity [3H]rauwolscine binding sites per cell (corresponding to 30-60 pmol/mg of protein) were obtained. The high expression level combined with relative ease and low cost of scaling-up make yeast a promising alternative to mammalian cells for the production of adrenergic and other G-protein-coupled receptors for structural studies.
Sizmann
D
D
VTT Biotechnology and Food Research, Espoo, Finland.
Kuusinen
H
H
Keränen
S
S
Lomasney
J
J
Caron
M G
MG
Lefkowitz
R J
RJ
Keinänen
K
K
D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D005285:Fermentation; D005822:Genetic Vectors; D006801:Humans; D010646:Phentolamine / Q000494:pharmacology; D015202:Protein Engineering; D011941:Receptors, Adrenergic / Q000096:biosynthesis* / Q000235:genetics*; D018341:Receptors, Adrenergic, alpha-2 / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development
D000317:Adrenergic alpha-Antagonists; D018076:DNA, Complementary; D011941:Receptors, Adrenergic; D018341:Receptors, Adrenergic, alpha-2; D018343:Receptors, Adrenergic, beta-2; D010646:Phentolamine