重组人死亡受体6胞外结构域的制备及与淀粉样前体蛋白N端片段相互作用鉴定

doi:10.13523/j.cb.20160201

中国生物工程杂志

2016, Vol. 36

Issue (2): 1-6 DOI: 10.13523/j.cb.20160201

研究报告

重组人死亡受体6胞外结构域的制备及与淀粉样前体蛋白N端片段相互作用鉴定

牛纯青¹, 高香¹, 罗金华², 李伟¹, 刘秋萍¹, 陈韵¹, 刘堰¹

1. 西南大学生命科学学院重庆 400715;
2. 重庆市生物技术研究所有限责任公司重庆 401121

Preparation of Recombinant Human Death Receptor 6 Ectodomain and Its Interaction with a Cleaved Amino-terminal Fragment of Human Amyloid Precursor Protein

NIU Chun-qing¹, GAO Xiang¹, LUO Jin-hua², LI Wei¹, LIU Qiu-ping¹, CHEN Yun¹, LIU Yan¹

1. School of Life Science, Southwest University, Chongqing 400715, China;
2. Chongqing Research Institute of Biotechnology Co., Ltd, Chongqing 401121, China

全文: PDF HTML

摘要：

人淀粉样前体蛋白氨基端切割片段(a cleaved amino-terminal fragment of amyloid precursor protein,N-APP)结合死亡受体6(Death receptor-6,DR6),会触发神经元和轴突依赖天冬氨酸特异性半胱氨酸蛋白酶的自我毁灭过程,从而导致阿尔茨海默病(Alzheimer's disease,AD)的发生。为了在分子水平上研究N-APP-DR6诱导神经元退化途径,制备大量纯化的重组DR6胞外结构域以及鉴定NAPP和DR6胞外结构域的结合位点是关键。从甲醇毕赤酵母中成功表达并纯化了重组DR6胞外域(aa42-349),得率高达90 mg/L。为了验证DR6和NAPP的相互作用关系,构建谷胱甘肽转移酶-死亡受体6(GST-DR6)(aa42-349)融合蛋白原核表达质粒,转化大肠杆菌(Escherichia coli,E.coli)BL21,表达融合蛋白GST-DR6(aa42-349),同时纯化得到DR6的配体NAPP,GST pull-down结果显示DR6与NAPP能够在细胞外发生相互作用。

关键词： 阿尔茨海默病; 淀粉样前体蛋白; GST pull down; 死亡受体-6; 毕赤酵母

Abstract:

The cleaved amino-terminal fragment of human amyloid precursor protein(N-APP) binds death receptor 6(DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer's disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, producing abundant and purified recombinant DR6 ectodomain and identification binding sites of NAPP and DR6 ectodomain are critical. Here, the recombinant DR6 ectodomain from the methylotrophic yeast Pichia pastoris were successfully expressed and purified with a high yield of 90 mg/L. In order to identify the protein-protein interaction between DR6 and NAPP in vitro, prokaryotic expression plasmid pGST-DR6(aa42-349) was constructed, and transformed into E. coli, the expression of fusion protein GST-DR6 was achieved by induction. Meanwhile, NAPP was purified. GST pull-down assay showed that DR6 and NAPP could interact with each other in vitro.

Key words: Amyloid precursor protein GST pull down Death receptor-6 Alzheimer' disease Pichia pastoris

收稿日期: 2015-09-18 出版日期: 2015-12-01

ZTFLH:

Q819

基金资助:

重庆市自然科学基金(CSTC.2011BB0146)资助项目

通讯作者: 刘堰 E-mail: liuyan@swu.edu.cn

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引用本文:

牛纯青, 高香, 罗金华, 李伟, 刘秋萍, 陈韵, 刘堰. 重组人死亡受体6胞外结构域的制备及与淀粉样前体蛋白N端片段相互作用鉴定[J]. 中国生物工程杂志, 2016, 36(2): 1-6.

NIU Chun-qing, GAO Xiang, LUO Jin-hua, LI Wei, LIU Qiu-ping, CHEN Yun, LIU Yan. Preparation of Recombinant Human Death Receptor 6 Ectodomain and Its Interaction with a Cleaved Amino-terminal Fragment of Human Amyloid Precursor Protein. China Biotechnology, 2016, 36(2): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160201 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I2/1

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