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

生物技术通报 ›› 2015, Vol. 31 ›› Issue (8): 88-93.doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.013

拟南芥SPX1蛋白原核表达及纯化分析

胡涛¹, 安艳¹, 吕群丹², 徐英武¹

1. 浙江农林大学亚热带森林培育国家重点实验室培育基地,临安 311300; 2. 浙江大学生命科学学院,杭州 310058

收稿日期:2014-12-12 出版日期:2015-08-21 发布日期:2015-08-22
作者简介:胡涛,男,硕士研究生,研究方向：生物物理;E-mail：462109237@qq.com
基金资助:
国家自然科学基金项目（31270715）,浙江农林大学科研发展基金项目（2010FR072）

Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana

Hu Tao¹, An Yan¹, Lü Qundan², Xu Yingwu¹

1. The Nurturing Station for the State Key Laboratory of Subtropical Silviculture,Zhejiang A & F University,Lin’an 311300; 2. College of Life Science,Zhejiang University,Hangzhou 310058

Received:2014-12-12 Published:2015-08-21 Online:2015-08-22

摘要/Abstract

摘要：

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。

关键词: SPX结构域, 原核表达, 蛋白纯化, 拟南芥

Abstract:

The proteins containing SPX domain exist widely in eukaryotes, and the functions of this kind of proteins are not clear yet, however some of them are involved in phosphorus signaling and some in iron signaling. SPX proteins in Arabidopsis thaliana can be divided into 4 families. AtSPX1 used in this paper belongs to the family containing only SPX domain, while other 3 families containing extra gene sequences. Phylogenetic analysis showed that amino acid encoded by AtSPX1 was close with dicots in sequence, but distant from monocots. In order to reveal the relationship between structural property and biological function, we carried out solubility expression experiments of the proteins in vitro, constructed prokaryotic expression vector in vitro, and had the high soluble expression of the protein in Escherichia coli’s cells. The expressed His-tag proteins allowed the purification more convenient, i.e, the inserted SUMO fusion protein tag could be digested by protease, and the target protein could be purified by ammonium sulfate precipitation. The further purification was completed using size exclusion chromatographic column, which yielded a profile corresponding to a monomeric AtSPX1 in solution. This work provided a strategy for the purification of AtSPX1 protein.

Key words: SPX domain, prokaryotic expression, protein purification, Arabidopsis thaliana

胡涛, 安艳, 吕群丹, 徐英武. 拟南芥SPX1蛋白原核表达及纯化分析[J]. 生物技术通报, 2015, 31(8): 88-93.

Hu Tao, An Yan, Lv Qundan, Xu Yingwu. Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2015, 31(8): 88-93.

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拟南芥SPX1蛋白原核表达及纯化分析

Prokaryotic Expression and Purification of AtSPX1 Protein in Arabidopsis thaliana

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