Production, purification and characterization of recombinant, full-length human claudin-1.
Production, purification and characterization of recombinant, full-length human claudin-1.
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DOI：
10.1371/journal.pone.0064517
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Bill RM
中科院分区：
文献类型：
--
作者：
Bonander N;Jamshad M;Oberthür D;Clare M;Barwell J;Hu K;Farquhar MJ;Stamataki Z;Harris HJ;Dierks K;Dafforn TR;Betzel C;McKeating JA;Bill RM
密蛋白超家族的跨膜结构域蛋白是细胞紧密连接的主要结构成分。一个家族成员claudin-1也与四跨膜蛋白CD81相关，作为受体复合物的一部分，该受体复合物对于肝脏的丙型肝炎病毒（HCV）感染至关重要。为了了解claudin-1/CD81关联的分子基础，我们之前生产并纯化了毫克量的功能性全长CD81，它结合可溶形式的HCV E2糖蛋白（sE2）。在这里，我们报告了claudin-1的生产、纯化和表征。酵母膜结合和去污剂提取的纯化claudin-1 均具有抗原性并可被特异性抗体识别。分析超速离心表明，用正辛基-β-d-吡喃葡萄糖苷提取可产生claudin-1的单分散二聚体池，而用profoldin-8或正癸基磷酸胆碱提取可产生claudin-1低聚物的动态混合物。两种形式均未与 sE2 结合，符合文献预期，而进一步的功能分析因发现将claudin-1掺入脂蛋白体而使其难以研究而受到阻碍。动态光散射表明，claudin-1 寡聚物在体外以 1:2 的确定摩尔比与 CD81 结合，并且胆固醇半琥珀酸酯的存在增强了复合物的形成。我们发现claudin-1影响脂蛋白体的特性，因此对该复合物进行生物学分析的尝试受到限制。我们得出的结论是，重组的、正确折叠的全长claudin-1可以在酵母膜中产生，它可以以不结合sE2的不同寡聚形式提取，并且动态制剂可以在没有任何其他细胞成分的情况下在体外与CD81形成特定的复合物。这些发现为 Claudin-1 单独和与 CD81 复合物的结构表征铺平了道路。
The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-β-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1∶2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.
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影响因子：
3.9
作者：
Bonander, Nicklas;Jamshad, Mohammed;Bill, Roslyn M.
通讯作者：
Bill, Roslyn M.
影响因子：
5.4
作者：
Farquhar, Michelle J.;Hu, Ke;McKeating, Jane A.
通讯作者：
McKeating, Jane A.
影响因子：
3.4
作者：
Harris, H. J.;Clerte, C.;McKeating, J. A.
通讯作者：
McKeating, J. A.
影响因子：
3.8
作者：
Dierks, Karsten;Meyer, Arne;Betzel, Christian
通讯作者：
Betzel, Christian
单个基因产物Claudin -1或-2，在成纤维细胞中重构紧密结合的连接链和新兵。
影响因子：
7.8
作者：
Furuse, M;Sasaki, H;Fujimoto, K;Tsukita, S
通讯作者：
Tsukita, S