Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

被引：31

作者：

Asada, Hidetsugu ^{[1
,2
]}

Uemura, Tomoko ^{[1
]}

Yurugi-Kobayashi, Takami ^{[1
,2
]}

Shiroishi, Mitsunori ^{[1
]}

Shimamura, Tatsuro ^{[1
,2
]}

Tsujimoto, Hirokazu ^{[1
]}

Ito, Keisuke ^{[3
]}

Sugawara, Taishi ^{[3
]}

Nakane, Takanori ^{[2
]}

Nomura, Norimichi ^{[1
,2
]}

Murata, Takeshi ^{[1
]}

Haga, Tatsuya ^{[4
]}

Iwata, So ^{[1
,2
,5
,6
]}

Kobayashi, Takuya ^{[1
,2
,5
]}

机构：

[1] JST, ERATO, Iwata Human Receptor Crystallog Project, Sakyo Ku, Kyoto 6068501, Japan

[2] Kyoto Univ, Dept Med Chem, Fac Med, Sakyo Ku, Kyoto 6068501, Japan

[3] Univ Tokyo, Grad Sch Agr & Life Sci, Dept Appl Biol Chem, Bunkyo Ku, Tokyo 1138657, Japan

[4] Gakushuin Univ, Fac Sci, Inst Biomol Sci, Toshima Ku, Tokyo 1718588, Japan

[5] RIKEN Genom Sci Ctr, Yokohama, Kanagawa 2300045, Japan

[6] Univ London Imperial Coll Sci Technol & Med, Div Mol Biosci, Membrane Prot Crystallog Grp, London SW7 2AZ, England

来源：

MICROBIAL CELL FACTORIES | 2011年 / 10卷

基金：

日本科学技术振兴机构;

关键词：

PROTEIN-COUPLED RECEPTOR; MUSCARINIC ACETYLCHOLINE-RECEPTOR; DROSOPHILA SCHNEIDER-2 CELLS; ADENOSINE A(2A) RECEPTOR; LARGE-SCALE PRODUCTION; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; LIGAND-BINDING; SACCHAROMYCES-CEREVISIAE; FUNCTIONAL EXPRESSION;

D O I：

10.1186/1475-2859-10-24

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 beta(2) adrenergic receptor, adenosine A(2a) 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 degrees 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 [H-3]-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. Conclusion: 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.

引用

页数：13

共 68 条

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