| 拟南芥Alpha-dioxygenase2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定 |
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| 引用本文： | 魏少华,杨宇衡,安瑞,单丽伟,范三红. 拟南芥Alpha-dioxygenase2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定[J]. 农业生物技术学报, 2011, 19(1). DOI: 10.3969/j.issn.1674-7968.2011.01.007 |
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| 作者姓名： | 魏少华 杨宇衡 安瑞 单丽伟 范三红 |
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| 作者单位： | 1. 西北农林科技大学生命科学学院,杨凌,712100
2. 西北农林科技大学生命科学学院,杨凌712100;陕西省农业分子生物学重点实验室,杨凌712100 |
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| 基金项目： | 国家自然科学基金(No.30300222); 西北农林科技大学青年科学基金(No.QN2009070)资助 |
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| 摘 要： | 为了获得具有生物活性的拟南芥(Arabidopsis thaliana)alpha-dioxygenase2(AtDOX2),将其对应基因AtDOX2编码区克隆到酵母表达载体pPIC9k中,获得重组表达载体pPIC9k-AtDOX2,将线性化的重组载体电击转化入毕赤酵母(Pichia pastoris)表达菌株GS115,经G418筛选、PCR鉴定和甲醇诱导时间优化,获得重组AtDOX2的高效表达菌株GS115/pPIC9k-AtDOX2。SDS-PAGE分析结果显示,0.5%甲醇诱导96h重组蛋白表达量最高,其表达量占胞外总蛋白的15%。重组AtDOX2的表观分子量约为70kD,经Ni-NTA柱亲和层析可获得纯度大于80%的重组蛋白。2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)法测定结果表明重组蛋白具有过氧化物酶活性,且其活性受Ca2+和Mg2+激活,受EDTA、咪唑和Mn2+抑制;2,4-二硝基苯肼(2,4-DNP)法测定结果显示,重组AtDOX2具有双加氧酶活性,Ca2+对其双加氧酶活性也有激活作用。结果说明利用酵母表达系统获得...
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| 关 键 词： | 拟南芥 AtDOX2 毕赤酵母 过氧化物酶 α-双加氧酶 |
Expression, Purification and Activity Identification of Arabidopsis thaliana Alpha-dioxygenase 2(AtDOX2)in Pichia pastoris |
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| Wei Shaohua,Yang Yuheng,An Rui,Shan Liwei,Fan Sanhong. Expression, Purification and Activity Identification of Arabidopsis thaliana Alpha-dioxygenase 2(AtDOX2)in Pichia pastoris[J]. Journal of Agricultural Biotechnology, 2011, 19(1). DOI: 10.3969/j.issn.1674-7968.2011.01.007 |
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| Authors: | Wei Shaohua Yang Yuheng An Rui Shan Liwei Fan Sanhong |
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| Affiliation: | Wei Shaohua1 Yang Yuheng1 An Rui1 Shan Liwei1 Fan Sanhong1,2 1 College of Life Sciences,Northwest Agriculture and Forestry University,Yangling 712100,China,2 Key Laboratory of Agriculture Molecular Biology of Shaanxi Province |
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| Abstract: | In order to obtain active Arabidopsis thaliana alpha-dioxygenase 2 (AtDOX2),the encoding sequence of AtDOX2 was cloned into pPIC9k to obtain pPIC9k-AtDOX2. The recombinant vector was linearized and electrophorated into Pichia pastoris strain GS115. After G418 selection,PCR analysis and optimization of methanol inducing time,the high level expression strain of GS115/pPIC9k-AtDOX2 was obtained. SDS-PAGE analysis revealed that the expression level of recombinant protein reached to top at 96 h after inducing by... |
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| Keywords: | AtDOX2 |
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