title: Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.
abstract: BACKGROUND
  Various protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.
  
  
  RESULTS
  The ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.
  
  
  CONCLUSIONS
  Compared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.
authors.affiliation: Iwata Human Receptor Crystallography project, ERATO, JST, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Tomoko
authors.initials: T
authors.lastname: Uemura
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Tsujimoto
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Takanori
authors.initials: T
authors.lastname: Nakane
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D043585:Receptor, Muscarinic M2 / Q000235:genetics* / Q000378:metabolism*; D018411:Spodoptera
chemical_list: C487078:CHRM2 protein, human; D043585:Receptor, Muscarinic M2