High-yield expression, reconstitution and structure of the recombinant, fully functional glutamate transporter GLT-1 from Rattus norvegicus
High-yield expression, reconstitution and structure of the recombinant, fully functional glutamate transporter GLT-1 from Rattus norvegicus
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DOI：
10.1016/j.jmb.2005.06.036
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发表时间：
2005-08-19
影响因子：
5.6
通讯作者：
Kühlbrandt, W
中科院分区：
文献类型：
--
作者：
Raunser, S;Haase, W;Kühlbrandt, W
利用Semliki Forest病毒表达系统在BHK细胞中高效表达了褐家鼠谷氨酸转运蛋白GLT-1。携带GLT-1基因的病毒颗粒感染的BHK细胞表现出30倍增加的天冬氨酸摄取相比，对照细胞。通过标记底物与膜制备物的结合测定GLT-1的表达水平约为3.5 × 10（6）个功能性转运蛋白/细胞，或61 pmol GLT-1/mg膜蛋白。通过Ni 2 +-NTA亲和层析纯化His标记的蛋白质使得能够常规生产和纯化毫克量的全功能转运蛋白。重组蛋白的表观分子量为73 kDa和55 kDa，分别对应于糖基化和非糖基化形式。两种形式在凝集素柱上分离并重构为脂质体后均具有活性。糖基化和非糖基化GLT-1以相同的效率转运到质膜。我们的研究结果表明，N-糖基化不影响GLT-1的运输或运输活性。通过电子显微镜和单颗粒重构确定了GLT-1的低分辨率结构。(c)2005爱思唯尔有限公司保留所有权利。
The glutamate transporter GLT-1 from Rattus norvegicus was expressed at high level in BHK cells using the Semliki Forest virus expression system. BHK cells infected with viral particles carrying the GLT-1 gene exhibited 30-fold increased aspartate uptake compared to control cells. The expression level of GLT-1 as determined by binding of labelled substrate to membrane preparations was about 3.5 X 10(6) functional transporters per cell, or 61 pmol GLT-1 per milligram of membrane protein. Purification of the His-tagged protein by Ni2+-NTA affinity chromatography enabled the routine production and purification of milligram quantities of fully functional transporter. Transport activity required reducing conditions and the addition of extra lipid throughout the purification.The apparent molecular mass of the recombinant transporter was 73 kDa or 55 kDa, corresponding to the glycosylated and non-glycosylated form, respectively. Both forms were active upon separation on a lectin column and reconstitution into liposomes. Glycosylated and non-glycosylated GLT-1 were transported to the plasma membrane with equal efficiency. Our results show that N-glycosylation does not affect the trafficking or the transport activity of GLT-1. The low-resolution structure of GLT-1 was determined by electron microscopy and single particle reconstruction. (c) 2005 Elsevier Ltd. All rights reserved.