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Expression, Purification and Characterization of C-FADD

ISSN号：1672-7681
期刊名称：Cellular & Molecular Immunology
时间：0
页码：295-301
语言：英文
分类：Q255[生物学—细胞生物学] V448.23[航空宇航科学与技术—飞行器设计;航空宇航科学技术]
作者机构：[1]The State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, College of Life Sciences, Nanjing University, Nanjing 210093, China, [2]Changzhou High-Tech Research Institute of Nanjing University, Changzhou213164, China, [3]State Key Laboratory of Pharmaceutical Biotechnology and Jiangsu Center of Hepatobiliary Diseases, College of Life Sciences, Nanjing University, Nanjing 210093, China
相关基金：Acknowledgements The authors are grateful to grants from the Chinese National Nature Sciences Foundation （30425009, 30330530, 3073003 0, 30600320, 30821006） and Jiangsu Provincial Department of Health.
相关项目：天然药源分子及其新作用特点

关键词：纯化, 细胞凋亡, 表征, 末端区域, 死亡受体, 细胞周期, 功能关系, 大肠杆菌, C-FADD, expression, purification, monomer

中文摘要：

FADD 是在死亡的一个重要 proapoptotic 适配器导致受体的 apoptosis。最近， FADD 被发现了参予许多 non-apoptotic 过程，例如开发，房间周期前进和幸存。它的 non-apoptotic 活动被在 C 终端区域定位的丝氨酸残余的 phosphorylated 地位调整，从 proapoptotic 函数不同的域联系了 DED 和 DD 域。由于在自然 FADD 的表示和结晶化的困难，然而，迄今为止，所有 FADD 变体的分子的结构没包含 C 终端区域。阐明 C 终端区域的结构功能关系，我们需要获得 FADD 变体那个包含的 C 终端区域。在这研究，包含 DD 领域的鼠标 FADD (80-205 ) 和 C 终端区域，指定了为 C-FADD，在 E 被表示。有在 N 终点的他的标签的 coli 并且由 Ni2+ 亲密关系层析净化了。净化的蛋白质在 glutaraldehyde cross-linking 分析作为同质的单体存在并且在 CD (圆形的二色性) 展出了一个典型螺旋系列试金。在 vitro 他的标签，下拉试金证明净化的 C-FADD 拥有了 CK 为它的 non-apoptotic 功能重要的我有约束力的活动。

英文摘要：

FADD is an important proapoptotic adaptor in death receptor-induced apoptosis. Recently, FADD has been found to participate in a variety of non-apoptotic processes, such as development, cell cycle progression and survival. Its non-apoptotic activities were regulated by the phosphorylated status of the serine residue located at the C-terminal region, a domain distinct from the proapoptotic function related DED and DD domains. However, due to the difficulties in expression and crystallization of natural FADD, by far the molecuiar structures of all FADD variants did not contain the C-terminal region. To elucidate the structure-function relationship of C-terminal region, we need to obtain a FADD variant that containing C-terminal region. In this study, mouse FADD （80-205） containing DD domain and C-terminal region, designated as C-FADD, was expressed in E. coli with His-tag at the N-terminus and purified by Ni^2＋ affinity chromatography. The purified protein existed as a homogenous monomer in glutaraldehyde cross-linking analysis and exhibited a typical a-helix spectrum in CD （circular dichroism） assay. In vitro His-tag pull-down assay demonstrated that the purified C-FADD possessed the CK Ia-binding activity which was important for its non-apoptotic function.

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