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

Asada H, Uemura T, Yurugi-Kobayashi T, Shiroishi M, Shimamura T, Tsujimoto H, Ito K, Sugawara T, Nakane T, Nomura N, Murata T, Haga T, Iwata S, Kobayashi T - Microb. Cell Fact. (2011)

Bottom Line: There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Iwata Human Receptor Crystallography project, ERATO, JST, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

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.

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.

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Comparison of culture conditions in P. pastoris and Sf9 insect cells. In P. pastoris, cell density (A), methanol concentration (B), and pH (C) were measured until 5 days after methanol induction. Cell density was estimated from OD600. Methanol concentration was monitored before and after methanol addition using a methanol sensor system (RAVEN Biotech Inc.). In Sf9 insect cells, cell density (D), glucose concentration (E), and pH (F) were measured during the period from preculture (4 days) to baculovirus infection (2 days). Cells were stained with 0.25% trypan blue in PBS (-) and living cells were counted with a hemocytometer.
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Figure 2: Comparison of culture conditions in P. pastoris and Sf9 insect cells. In P. pastoris, cell density (A), methanol concentration (B), and pH (C) were measured until 5 days after methanol induction. Cell density was estimated from OD600. Methanol concentration was monitored before and after methanol addition using a methanol sensor system (RAVEN Biotech Inc.). In Sf9 insect cells, cell density (D), glucose concentration (E), and pH (F) were measured during the period from preculture (4 days) to baculovirus infection (2 days). Cells were stained with 0.25% trypan blue in PBS (-) and living cells were counted with a hemocytometer.

Mentions: Culture conditions such as cell density, nutrient metabolism and pH, which contribute to optimal recombinant protein production, were compared between P. pastoris and insect cells. P. pastoris transformants were inoculated and grown in BMGY medium, in which glycerol was the sole carbon source, overnight or until an OD600 of 2-6 was reached. The cells were then harvested by centrifugation. To induce expression of the protein of interest by methanol, the cell pellet was resuspended to an OD600 of 1.0, corresponding to 5 × 107 cells/ml in BMMY medium (0.5% methanol). The cell density increased up to 2.4 ± 0.3 × 109 cells/ml in the baffled shake flask until 3 days after methanol induction of the recombinant protein (Figure 2A). Methanol was added to a final concentration of 1% every 24 hr during the induction phase, maintaining the total concentration between 0.5% and 1.5% to minimize toxicity (Figure 2B). The pH in the induction medium gradually decreased, requiring 5 days for a reduction from 7.0 to 6.5 ± 0.2 (Figure 2C).


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

Asada H, Uemura T, Yurugi-Kobayashi T, Shiroishi M, Shimamura T, Tsujimoto H, Ito K, Sugawara T, Nakane T, Nomura N, Murata T, Haga T, Iwata S, Kobayashi T - Microb. Cell Fact. (2011)

Comparison of culture conditions in P. pastoris and Sf9 insect cells. In P. pastoris, cell density (A), methanol concentration (B), and pH (C) were measured until 5 days after methanol induction. Cell density was estimated from OD600. Methanol concentration was monitored before and after methanol addition using a methanol sensor system (RAVEN Biotech Inc.). In Sf9 insect cells, cell density (D), glucose concentration (E), and pH (F) were measured during the period from preculture (4 days) to baculovirus infection (2 days). Cells were stained with 0.25% trypan blue in PBS (-) and living cells were counted with a hemocytometer.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3094209&req=5

Figure 2: Comparison of culture conditions in P. pastoris and Sf9 insect cells. In P. pastoris, cell density (A), methanol concentration (B), and pH (C) were measured until 5 days after methanol induction. Cell density was estimated from OD600. Methanol concentration was monitored before and after methanol addition using a methanol sensor system (RAVEN Biotech Inc.). In Sf9 insect cells, cell density (D), glucose concentration (E), and pH (F) were measured during the period from preculture (4 days) to baculovirus infection (2 days). Cells were stained with 0.25% trypan blue in PBS (-) and living cells were counted with a hemocytometer.
Mentions: Culture conditions such as cell density, nutrient metabolism and pH, which contribute to optimal recombinant protein production, were compared between P. pastoris and insect cells. P. pastoris transformants were inoculated and grown in BMGY medium, in which glycerol was the sole carbon source, overnight or until an OD600 of 2-6 was reached. The cells were then harvested by centrifugation. To induce expression of the protein of interest by methanol, the cell pellet was resuspended to an OD600 of 1.0, corresponding to 5 × 107 cells/ml in BMMY medium (0.5% methanol). The cell density increased up to 2.4 ± 0.3 × 109 cells/ml in the baffled shake flask until 3 days after methanol induction of the recombinant protein (Figure 2A). Methanol was added to a final concentration of 1% every 24 hr during the induction phase, maintaining the total concentration between 0.5% and 1.5% to minimize toxicity (Figure 2B). The pH in the induction medium gradually decreased, requiring 5 days for a reduction from 7.0 to 6.5 ± 0.2 (Figure 2C).

Bottom Line: There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Iwata Human Receptor Crystallography project, ERATO, JST, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.

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.

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.

Show MeSH
Related in: MedlinePlus