Mondal Samsuzzoha, James Honey Priya, Milano Francesco, Jin Rui, Baumgart Tobias
Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, United States.
Bio Protoc. 2023 Jun 20;13(12):e4699. doi: 10.21769/BioProtoc.4699.
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.
Bin/Amphiphysin/Rvs（BAR）蛋白是已知的内吞作用过程中的经典膜曲率生成器。发动蛋白是N-BAR亚家族蛋白的成员，在BAR结构域的N端含有一个特征性的两亲序列，参与网格蛋白介导的内吞作用。全长发动蛋白包含一个约400个氨基酸长的无序连接子，连接N-BAR结构域和C端Src同源3（SH3）结构域。我们表达并纯化了重组发动蛋白及其N-BAR结构域，并带有N端谷胱甘肽-S-转移酶（GST）标签。GST标签允许使用亲和色谱法提取目标蛋白，并在随后的蛋白酶处理和离子交换色谱步骤中去除。对于N-BAR结构域，发现GST标签的切割会导致沉淀。通过向蛋白质纯化缓冲液中添加甘油，可以将这个问题最小化。在最后一步中，尺寸排阻色谱法去除任何潜在的寡聚体。该方案也已成功用于纯化其他N-BAR蛋白，如内吞蛋白、Bin1及其相应的BAR结构域。图形概述。
