Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.
Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis.
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DOI：
10.1186/1475-2859-10-24
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发表时间：
2011-04-22
影响因子：
6.4
通讯作者：
Kobayashi T
中科院分区：
文献类型：
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作者：
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
各种蛋白质表达系统，如大肠杆菌 (E. coli)、酿酒酵母 (S. cerevisiae)、巴斯德毕赤酵母 (P. Pastoris)、昆虫细胞和哺乳动物细胞系，已被开发用于合成 G 蛋白偶联受体 (GPCR) 以进行结构研究。最近，利用昆虫细胞表达系统成功确定了四种重组人GPCR的晶体结构，即β2肾上腺素受体、腺苷A2a受体、CXCR4和多巴胺D3受体。昆虫细胞中表达的 GPCR 被认为经历了类似哺乳动物的转录后修饰，并且具有与哺乳动物相似的功能特性。 GPCR 的晶体结构尚未使用酵母表达系统解析。在本研究中，开发了人毒蕈碱乙酰胆碱受体M2亚型（CHRM2）的毕赤酵母和昆虫细胞表达系统，并比较了两种表达系统合成的CHRM2的数量和质量，以用于结构研究。在毕赤酵母中表达 CHRM2 的理想条件是在 20°C、pH 7.0 的缓冲液中表达 60 小时。通过使用[ 3 H]-苯苯甲酸奎宁环酯(QNB)的结合测定测定，所表达的CHRM2的比活性为28.9 pmol/mg膜蛋白。尽管毕赤酵母产生的蛋白质的比活性低于Sf9昆虫细胞，但毕赤酵母中CHRM2的产量比Sf9昆虫细胞高2倍，因为毕赤酵母是在高细胞密度下培养的。毕赤酵母中 QNB 的解离常数 (Kd) 为 101.14 ± 15.07 pM，与 Sf9 昆虫细胞中的解离常数 (Kd) 相似 (86.23 ± 8.57 pM)。巴斯德毕赤酵母和Sf9昆虫细胞之间CHRM2对QNB的结合亲和力没有差异。与昆虫细胞相比，毕赤酵母更容易处理，可以以更低的成本生长，并且可以更快地大规模表达。酵母、毕赤酵母和昆虫细胞都是 GPCR 的有效表达系统。本研究的结果强烈表明毕赤酵母中的蛋白质表达可应用于 GPCR 的结构和生化研究。
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. 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. 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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影响因子：
4
作者：
Alkhalfioui, Fatima;Magnin, Thierry;Wagner, Renaud
通讯作者：
Wagner, Renaud
影响因子：
3.6
作者：
Furukawa, H;Hamada, T;Ishiguro, M
通讯作者：
Ishiguro, M
影响因子：
16.2
作者：
BONNER, TI;YOUNG, AC;BUCKLEY, NJ
通讯作者：
BUCKLEY, NJ
影响因子：
2.2
作者：
Brillet, Karl;Perret, Benedicte G.;Wagner, Renaud
通讯作者：
Wagner, Renaud
DOI：
10.1007/978-1-60761-344-2_8
发表时间：
2010-01-01
期刊：
HETEROLOGOUS EXPRESSION OF MEMBRANE PROTEINS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Brillet, Karl;Pereira, Carlos A.;Wagner, Renaud
通讯作者：
Wagner, Renaud