Purification of human β2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris
Purification of human β2-adrenergic receptor expressed in methylotrophic yeast Pichia pastoris
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DOI：
10.1093/jb/mvj211
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发表时间：
2006-12-01
影响因子：
2.7
通讯作者：
Satow, Yoshinori
中科院分区：
文献类型：
--
作者：
Noguchi, Shuji;Satow, Yoshinori
人β 2肾上腺素能受体是一种G蛋白偶联受体，具有7个跨膜螺旋，在肺和心血管疾病的药物靶向中具有重要作用。为了获得高表达量的毕赤酵母转化子，设计了具有优化密码子使用的N-末端组氨酸标记的基因构建体。将构建体插入pPIC 9载体中，然后电穿孔到SMD 1168菌株中。获得的最高表达水平为约4 mg/升培养液。膜组分中受体对CGP-12177拮抗剂的解离常数为1.2 nM。受体用蔗糖单月桂酸酯溶解，并用一系列色谱步骤纯化，包括阴离子交换，Ni-琼脂糖，alprenolol-琼脂糖和羟基磷灰石柱。该受体是不均一糖基化的，显示约70-90 kDa的宽SDS-PAGE条带。经糖苷内切酶处理后，受体出现为约45 kDa的单一条带，并进一步用羟基磷灰石和凝胶过滤柱纯化。受体在凝胶过滤洗脱体积处洗脱为尖锐峰，对应于117 kDa的分子量。用SDS-PAGE分析，如此纯化的糖修剪受体是均一的，显示出对CGP-12177拮抗剂的解离常数为4.7 nM，并且适合于结晶实验。
Human beta(2)-adrenergic receptor is a G-protein-coupled receptor with seven transmembrane helices, and is important in pharmaceutical targeting on pulmonary and cardiovascular diseases. N-terminal histidine-tagged gene constructs with optimized codon usage were designed so as to obtain Pichia pastoris transformants with a high expression level. The constructs were inserted into the pPIC9 vector, and then electroporated into the SMD1168 strains. The highest expression level obtained was about 4 mg/liter-culture broth. The dissociation constant of the receptor in the membrane fraction was 1.2 nM toward CGP-12177 antagonist. The receptor was solubilized with sucrose monolaurate and purified with a series of chromatography steps including anion-exchange, Ni-Sepharose, alprenolol-Agarose, and hydroxyapatite columns. The receptor was heterogeneously glycosylated, showing broad SDS-PAGE bands around 70-90 kDa. After endoglycosidase treatment, the receptor appeared as a single band around 45 kDa, and was further purified with hydroxyapatite and gel-filtration columns. The receptor was eluted as a sharp peak at the gel-filtration elution volume corresponding to a molecular mass of 117 kDa. The saccharide-trimmed receptor thus purified is homogeneous as analyzed with SDS-PAGE, shows the dissociation constant of 4.7 nM toward CGP-12177 antagonist, and is suitable for crystallization experiments.