Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris -: Importance of correct processing of the N-terminus
Biochemical and mass spectrometric characterization of the human CB2 cannabinoid receptor expressed in Pichia pastoris -: Importance of correct processing of the N-terminus
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DOI：
10.1016/j.pep.2007.03.018
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发表时间：
2007-10-01
影响因子：
1.6
通讯作者：
Song, Zhao-Hui
中科院分区：
文献类型：
--
作者：
Zhang, Rundong;Kim, Tae-Kang;Song, Zhao-Hui
本研究旨在优化甲基营养酵母巴斯德毕赤酵母（P. Pastoris）中人类 CB2 大麻素受体的表达。在蛋白质印迹中观察到表达的 CB2 蛋白的两个主要种类，即与标记 CB2 的计算分子量相匹配的 42 kDa 带和 52/55 kDa 双联体。在诱导过程中用 N-糖苷酶 F 处理膜或在培养基中加入衣霉素会导致 55 kDa 条带消失，但 52 kDa 条带不会消失，这表明 55 kDa 带中额外的 3 kDa 是由于 N-糖基化造成的，但 52 kDa 带中额外的 10 kDa 不是由于 N-糖基化造成的。与 52/55 kDa 双联体相比，抗 FLAG M1 抗体对 42 kDa 条带的偏好要高得多，并且通过 CNBr 消化 52/55 双联体产生了抗 FLAG M2 抗体识别的 10 kDa 片段。这些数据有力地支持了以下假设：分子量增加 10 kDa 是由于未经处理的 a 因子序列所致。通过使用胃蛋白酶/胰凝乳蛋白酶消化和 LC/MS/MS 方法找到 CB2 受体 N 端 a 因子片段的几个肽序列，进一步验证了这一结论。重要的是，未加工的α因子被发现与较差的配体结合有关。此外，控制 CB2 蛋白表达水平对于最大限度地减少未加工的 a 因子序列的存在至关重要。从这项研究中获得的信息不仅有助于 CB2 受体的正确表达，而且有助于 GPCR 家族其他成员在毕赤酵母中的正确表达。 (C) 2007 Elsevier Inc. 保留所有权利。
This study was conducted to optimize the expression of human CB2 cannabinoid receptors in methylotrophic yeast Pichia pastoris (P. pastoris). Two major species of expressed CB2 proteins were seen on Western blot, i.e., a 42 kDa band which matches the calculated molecular weight for tagged CB2, and a 52/55 kDa doublet. Treatment of membranes with N-glycosidase F or inclusion of tunicamycin in the culture medium during induction resulted in the disappearance of the 55 kDa, but not the 52 kDa band, suggesting that the 3 kDa extra in the 55 kDa band is due to N-glycosylation, but the 10 kDa extra in the 52 kDa band is not due to N-glycosylation. Anti-FLAG M1 antibody had a much higher preference for the 42 kDa band over the 52/55 kDa doublet, and a 10 kDa fragment recognized by anti-FLAG M2 antibody was generated by CNBr digestion of the 52/55 doublet. These data strongly support the hypothesis that the 10 kDa increase in molecular weight was due to unprocessed a-factor sequence. This conclusion was further validated by finding several peptide sequences for a-factor fragments at the N-terminal of the CB2 receptor using pepsin/chymotrypsin digestion and LC/MS/MS approaches. Importantly, unprocessed a-factor was found to be associated with poor ligand binding. In addition, controlling the level of CB2 protein expression was found to be critical for minimizing the presence of unprocessed a-factor sequence. The information gained from this study should aid the proper expression of not only CB2 receptor but also other members of the GPCR family in P. pastoris. (C) 2007 Elsevier Inc. All rights reserved.