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| 长牡蛎电压门控钙离子通道β亚基的无标签亲和层析纯化 |
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刘凤华1,2,3, 王淏1,3, 贾乔雅1,3, 毕允晨1,2,3
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1.中国科学院海洋研究所 中国科学院海洋大科学研究中心, 中国科学院和山东省实验海洋生物学重点实验室, 山东 青岛 266071;2.中国科学院大学, 北京 100049;3.青岛海洋科学与技术试点国家实验室 海洋生物学与生物技术功能实验室, 山东 青岛 266237
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| 摘要: |
| 亲和层析是一种广泛使用的蛋白质分离纯化技术，常用的亲和标签包括谷胱甘肽巯基转移酶（GST）标签、麦芽糖结合蛋白（MBP）标签、多组氨酸（polyhistidine）标签等。然而，外源亲和标签可能会影响蛋白质的结构与功能，通常需要酶切去除，造成实验流程加长、产率降低。某些蛋白质具有与亲和标签相似的氨基酸序列特征，利用这种序列特征尝试无标签纯化可有效避免上述问题。本研究选取C端天然富含组氨酸残基的长牡蛎电压门控钙离子通道（VGCC）β亚基进行无标签亲和层析纯化，结果显示，氨基三乙酸镍（Ni-NTA）能够特异性地结合无标签的β亚基，且结合能力与多组氨酸标签相当。对β亚基C端组氨酸残基分组突变后发现，随着组氨酸残基突变的增多，各突变型β亚基与Ni-NTA的结合能力逐步下降。免疫印迹实验结果表明，β亚基C端（HG）7序列在针对多组氨酸标签的抗体识别中发挥关键作用。VGCC β亚基的这种天然序列特征也可应用于该膜蛋白复合体提取过程关键步骤的监测。综上，本研究对氨基酸序列中具有亲和标签特征的蛋白质进行了无标签亲和层析纯化，为具有相似特征蛋白质的分离纯化提供了参考。 |
| 关键词: 电压门控钙离子通道 β亚基 蛋白纯化 亲和层析 多组氨酸 |
| DOI：10.11759/hykx20211231003 |
| 分类号:Q816 |
| 基金项目:青岛海洋科学与技术试点国家实验室海洋生物学与生物技术功能实验室引进人才支持计划项目（YJ2019NO03） |
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| Purification of voltage-gated calcium channel β subunit from Pacific oyster with tag-free affinity chromatography |
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LIU Feng-hua1,2,3, WANG Hao1,3, JIA Qiao-ya1,3, BI Yun-chen1,2,3
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1.CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.University of Chinese Academy of Sciences, Beijing 100049, China;3.Marine Biology and Biotechnology Laboratory, Pilot National Laboratory for Marine Science and Technology(Qingdao), Qingdao 266237, China
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| Abstract: |
| Affinity chromatography is widely used in protein extraction and purification. Common affinity tags include the glutathione sulfhydryl transferase (GST) tag, the maltose-binding protein (MBP) tag, and the polyhistidine tag. However, the attachment of an exogenous affinity tag could affect the structure and function of the target protein, thus usually further removed via enzyme digestion, causing longer procedures and lower yields. Certain amino acid sequences, naturally found in some proteins, feature similarity to specific affinity tags. Tag-free purification based on this sequence feature may effectively avoid the mentioned problems. In this study, the voltage-gated calcium channel (VGCC) β subunit, which is naturally histidine-rich in the C-terminal, was purified with tag-free affinity chromatography. The results showed that Ni-NTA could specifically bind to the β subunit, and the binding ability is equivalent to that of the polyhistidine tag. Mutant β subunits showed gradual decreasing binding ability with Ni-NTA correlating to the number of mutated histidine residues. Western blot analysis with anti-His tag showed specific blotting of β subunit and the C-terminal (HG)7 sequence was key to the antibody recognition. This natural amino acid feature of the VGCC β subunit was also successfully employed as indication of the VGCC complex integrity in the key steps of protein extraction. In summary, tag-free affinity chromatography purification strategy is performed in this study by relying on the natural amino acid sequence similarity to affinity tags found in some proteins, providing a reference for extraction and purification of proteins with similar characteristics. |
| Key words: voltage-gated calcium channel β subunit protein purification affinity chromatography polyhistidine |