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首页> 外文期刊>Biological chemistry >Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification
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Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification

机译：将过表达的整合膜蛋白从宿主膜快速转移到可溶性脂质纳米盘中，无需事先纯化

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摘要

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.
机译：由于去污剂通常会影响其结构和/或活性，因此在双层环境中完整膜蛋白的结构和功能表征会受到去污剂增溶和后续纯化的要求的严重阻碍。在这里，我们描述了一种快速的程序，只需最少暴露于去污剂即可在纯化之前将过表达的整合膜蛋白直接组装成可溶性脂质纳米盘。以重组的带有组氨酸标签的视紫红质为例，可以从其宿主膜中快速提取出视紫红质，并将其直接组装成膜支架蛋白（MSP）纳米光盘。我们进一步证明，即使MSP也带有组氨酸标签，也可以利用固定化金属色谱法对视紫红质-纳米纤维进行部分纯化。在纯化的纳米圆盘级分中，视紫红质的回收率高达80％。可以使用单个Ni2 +亲和层析步骤从视紫红质纳米糖中去除超过95％的污染膜蛋白和带有组氨酸标签的MSP。此纯化水平足以用于功能研究。我们提供的证据表明，所获得的视紫红质-纳米纤维制剂在光化学和结合相关G蛋白的能力方面均具有充分的功能。

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