High yield cell-free production of integral membrane proteins without refolding or detergents.
High yield cell-free production of integral membrane proteins without refolding or detergents.
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DOI：
10.1016/j.bbamem.2008.01.023
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发表时间：
2008-05
期刊：
影响因子：
--
通讯作者：
Jessica J Wuu;J. Swartz
中科院分区：
文献类型：
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作者：
Jessica J Wuu;J. Swartz
整合膜蛋白作为关键的细胞组分，是重要的药物靶标。然而，生产膜蛋白的困难阻碍了结构和功能的研究。体内生产系统通常受到细胞毒性的限制，并且先前的体外方法需要使用去污剂或脂质溶液的非天然折叠途径。为了克服这些局限性，我们提出了一种改进的无细胞表达系统，该系统在不使用去污剂或重折叠步骤的情况下产生高产量的整合膜蛋白。我们的无细胞反应激活大肠杆菌衍生的细胞提取物进行转录和翻译。纯化E.大肠杆菌内膜囊泡提供膜结合组分和插入和折叠所需的脂质环境。使用该系统，我们成功地合成了两种复杂的整合膜转运蛋白，四环素泵（TetA）和甘露醇通透酶（MtlA），产量分别为570±50 μg/mL和130±30 μg/mL的囊泡相关蛋白。这些产率是典型体内浓度的高达400倍。这些蛋白质的插入和折叠通过蔗糖浮选、蛋白酶消化和活性测定来验证。而TetA有效地结合到囊泡膜中，超过三分之二的合成蛋白被插入，MtlA产量似乎受到膜相关伴侣蛋白浓度不足的限制。
Integral membrane proteins act as critical cellular components and are important drug targets. However, difficulties in producing membrane proteins have hampered investigations of structure and function. In vivo production systems are often limited by cell toxicity, and previous in vitro approaches have required unnatural folding pathways using detergents or lipid solutions. To overcome these limitations, we present an improved cell-free expression system which produces high yields of integral membrane proteins without the use of detergents or refolding steps. Our cell-free reaction activates an Escherichia coli-derived cell extract for transcription and translation. Purified E. coli inner membrane vesicles supply membrane-bound components and the lipid environment required for insertion and folding. Using this system, we demonstrated successful synthesis of two complex integral membrane transporters, the tetracycline pump (TetA) and mannitol permease (MtlA), in yields of 570±50 μg/mL and 130±30 μg/mL of vesicle-associated protein, respectively. These yields are up to 400 times typical in vivo concentrations. Insertion and folding of these proteins are verified by sucrose flotation, protease digestion, and activity assays. Whereas TetA incorporates efficiently into vesicle membranes with over two-thirds of the synthesized protein being inserted, MtlA yields appear to be limited by insufficient concentrations of a membrane-associated chaperone.