High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81.
High-level expression, single-step immunoaffinity purification and characterization of human tetraspanin membrane protein CD81.
复制标题
人四跨膜蛋白 CD81 的高水平表达、单步免疫亲和纯化和表征。
DOI：
10.1371/journal.pone.0002314
复制
发表时间：
2008-06-04
期刊：
影响因子：
3.7
通讯作者：
Khorana, H. Gobind
中科院分区：
文献类型：
--
作者：
Takayama, Hidehito;Chelikani, Prashen;Reeves, Philip J.;Zhang, Shuguang;Khorana, H. Gobind
对膜蛋白结构和功能的研究需要其以全功能形式的高水平表达和纯化。我们以前使用四环素诱导的稳定的哺乳动物细胞系，HEK 293 S-TetR，调节高水平表达的G蛋白偶联受体。我们在此报告成功地使用这种方法高水平表达从头寡聚DNA组装的人CD 81基因。CD 81是重要的四跨膜蛋白家族成员。最近被鉴定为丙型肝炎病毒包膜E2糖蛋白（HCV-E2）的推定受体。在这项研究中，我们使用一步rho-1D 4亲和纯化方法从HEK 293 S-TetR诱导的稳定细胞系中获得>95%的纯度。使用ELISA测定，我们确定纯化的CD 81受体对HCV-E2蛋白的亲和力为3.8±1.2 nM。使用荧光共聚焦显微镜，我们表明，诱导过表达的CD 81受体在HEK 293 S-TetR细胞正确地位于质膜上。我们证明了高水平表达的CD 81与有效的单步免疫亲和纯化的组合是一种有用的方法，用于获得大量的CD 81膜受体，适合于这种难以捉摸的四跨膜蛋白的详细结构分析。此外，这种简单的一步免疫亲和纯化膜蛋白的高纯度可以广泛用于其他膜蛋白的纯化，从而加速确定大量难以获得的膜蛋白的结构。
The study of membrane protein structure and function requires their high-level expression and purification in fully functional form. We previously used a tetracycline-inducible stable mammalian cell line, HEK293S-TetR, for regulated high-level expression of G-protein coupled receptors. We here report successfully using this method for high-level expression of de novo oligo-DNA assembled human CD81 gene. CD81 is a member of the vital tetraspanin membrane protein family. It has recently been identified as the putative receptor for the Hepatitis C Virus envelope E2 glycoprotein (HCV-E2). In this study we used a single-step rho-1D4-affinity purification method to obtain >95% purity from HEK293S-TetR-inducible stable cell lines. Using ELISA assay we determined that the affinity of the purified CD81 receptor for HCV-E2 protein is 3.8±1.2 nM. Using fluorescent confocal microscopy we showed that the inducibly overexpressed CD81 receptor in HEK293S-TetR cells is correctly located on the plasma membrane. We demonstrated that the combination of high-level expression of CD81 with efficient single-step immunoaffinity purification is a useful method for obtaining large quantities of CD81 membrane receptor suitable for detailed structural analyses of this elusive tetraspanin protein. Furthermore, this simple single-step immunoaffinity purification to high purity of membrane protein could be useful broadly for other membrane protein purifications, thus accelerating the determination of structures for large numbers of difficult-to-obtain membrane proteins.
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影响因子：
5.4
作者：
Petracca, R;Falugi, F;Grandi, G
通讯作者：
Grandi, G
DOI：
10.1073/pnas.212519199
发表时间：
2002-10-15
影响因子：
11.1
作者：
Reeves, PJ;Kim, JM;Khorana, HG
通讯作者：
Khorana, HG
影响因子：
8
作者：
Wallin, E;von Heijne, G
通讯作者：
von Heijne, G
影响因子：
8
作者：
Chelikani, Prashen;Reeves, Philip J.;Khorana, H. Gobind
通讯作者：
Khorana, H. Gobind
影响因子：
14.9
作者：
Hoover, DM;Lubkowski, J
通讯作者：
Lubkowski, J