Yeast Expression

Various yeasts representing lower eukaryotic organisms are commonly used for recombinant protein expression. For instance, Baker's yeast, Saccharomyces cere-visiae, has been subjected to GPCR expression. The yeast a-factor Ste2p was expressed with C-terminal FLAG and His6 tags at yields up to 1 mg.29 Purified Ste2p receptor could be reconstituted into artificial phospholipid vesicles. Nevertheless, restoration of ligand-binding activity required the presence of solubilized yeast membranes. In addition, the human dopamine D1A receptor was successfully expressed and purified with a His6 tag before reconstitution.30

Another yeast strain employed for recombinant protein expression is the fission yeast Schizosaccharomyces pombe, which has glycosylation patterns different from those of S. cerevisiae. Two types of S. pombe vectors have been engineered: for stable expression, a vector for chromosomal gene integration31 and for transient expression, an episomal vector.32 S. pombe is more similar to mammalian cells than other yeast strains possessing mammalian-like signal transduction systems.11 The human dopamine D2 receptor overexpressed in fission yeast was localized to the plasma membrane and the yields were three times higher than in S. cerevisiae.33

The Pichia pastoris methylotrophic yeast has become very attractive as a host for recombinant protein expression because of its capacity to perform many post-translational modifications including glycosylation, disulfide bond formation, and proteolytic processing.34 P. pastoris utilizes the methanol-regulated alcohol oxidase promoter I (AOX1) and the vector DNA can integrate as several copies in the host genome. Various GPCRs have been successfully expressed from these vectors.35 Three-fold enhanced expression of the mouse serotonin 5HT5A receptor (m5HT5AR) and the human p2 AR was obtained after fusion to the prepropeptide sequence of the S. cerevisiae a-factor. Addition of the antagonist alprenolol to the culture medium increased the binding activity for the p2 AR. A similar effect was seen for the m5HT5AR in the presence of yohimbine. Bmax values of 25 and 40 pmol/mg were obtained for p2 AR and m5-HT5AR, respectively.

The GPCRs expressed in P. pastoris showed pharmacological profiles similar to those observed in mammalian cells. Expression of 100 GPCRs in the MePNet structural genomics program (see Chapter 14) attained a success rate of >95% in P. pastoris. Expression optimization by temperature shifts, additives, and ligand supplements to culture media increased the Bmax values to >100 pmol/mg for certain GPCRs. Finally, another methylotrophic yeast strain, Hansenula polymorpha, was evaluated as a host for chemokine receptor CCR5 and CXCR4 expression, but the yields were much lower than for P. pastoris (unpublished results, Dr. Renaud Wagner, University of Strasbourg, France).

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