Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.
Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain.
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DOI：
10.21769/bioprotoc.4699
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发表时间：
2023-06-20
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影响因子：
0.8
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中科院分区：
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Bin/Amphiphyn/Rvs (BAR) 蛋白被称为胞吞过程中的经典膜曲率发生器。 Amphiphyn 是 N-BAR 蛋白质亚家族的成员，在 BAR 结构域的 N 末端含有特征性两亲序列，参与网格蛋白介导的内吞作用。全长两性蛋白含有约 400 个氨基酸长的无序接头，连接 N-BAR 结构域和 C 端 Src 同源 3 (SH3) 结构域。我们表达并纯化重组两性蛋白及其 N-BAR 结构域以及 N 端谷胱甘肽-S-转移酶 (GST) 标签。 GST 标签允许使用亲和层析提取感兴趣的蛋白质，并在随后的蛋白酶处理和离子交换层析步骤中去除。就 N-BAR 结构域而言，GST 标签的切割被发现会导致沉淀。通过向蛋白质纯化缓冲液中添加甘油可以最大限度地减少这个问题。在最后一步中，尺寸排阻色谱法去除任何潜在的低聚物质。该方案还已成功用于纯化其他 N-BAR 蛋白，例如内亲素、Bin1 及其相应的 BAR 结构域。 图形概览
Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains. Graphical overview