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拟南芥SPX1蛋白原核表达及纯化分析

DOI: 10.13560/j.cnki.biotech.bull.1985.2015.08.013, PP. 88-93

Keywords: SPX结构域,原核表达,蛋白纯化,拟南芥

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Abstract:

包含SPX结构域的蛋白在高等真核生物中广泛存在,这类蛋白的功能多数还不太清晰,但发现有些与磷信号相关,有些与铁信号相关。拟南芥中含有SPX结构域的蛋白可分为4个家族,本研究中的拟南芥SPX1(AtSPX1)属于一个只含有SPX结构域的蛋白组成的家族,其它家族成员还包含额外的基因序列。进化树分析表明,AtSPX1编码的氨基酸序列与双子叶植物具有较高的一致性,与单子叶植物进化距离较远。为了揭示AtSPX1蛋白的结构形态与其生物学功能之间的联系,开展了AtSPX1蛋白质体外可溶性表达实验,构建了原核体外表达载体,在大肠杆菌(E.coli)细胞中获得了该蛋白可溶性高表达。表达的蛋白包含有His标签方便了蛋白纯化,插入的SUMO融合蛋白标签可以通过蛋白酶切除,而目标蛋白通过硫酸铵沉淀实现了纯化。进一步分子筛层析分析表明AtSPX1以单体形式存在。实验结果提供了一套表达纯化AtSPX1蛋白的有效方案。

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