拟南芥Alpha-dioxygenase 2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定  

Expression,Purification and Activity Identification of Arabidopsis thaliana Alpha-dioxygenase 2 (AtDOX2) in Pichia pastoris

作　　者：魏少华[1] 杨宇衡[1] 安瑞[1] 单丽伟[1] 范三红[1,2] 

机构地区：[1]西北农林科技大学生命科学学院,杨凌712100 [2]陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2011年第1期51-56,共6页Journal of Agricultural Biotechnology

基　　金：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

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了具有活性的重组AtDOX2,AtDOX2同时具有过氧化物酶活性和双加氧酶活性。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 0.5% methanol and the target protein accounted for 15% of the extracellular total protein. The 70 kD recombinant AtDOX2 was purified by the Ni-NTA chromatography column,and the purity of recombinant protein was more than 80%. The peroxidase activity of the purified protein was tested by the 2,2＇-Azino-bis（3-ethylbenzothiazoline-6-sulfonic acid）（ABTS） assay,the result showed that the recombinant protein had peroxidase activity,and the peroxidase activity of AtDOX2 could be activated by Ca2＋ and Mg2＋ and inhibited by EDTA,imidazole and Mn2＋. The α-dioxygenase activity was analyzed by 2,4-dinitrophenyl hydraz（2,4-DNP） assay,the result showed that the recombinant protein had dioxygenase activity,and the α-dioxygenase activity of AtDOX2 could be activated by Ca2＋ too. Results indicate that the ecombinant AtDOX2 was expressed and purified by yeast system,and it acts as a dual functional enzyme of peroxidase and dioxygenase.

关 键 词：拟南芥 AtDOX2 毕赤酵母 过氧化物酶 α-双加氧酶 

分 类 号：Q786[生物学—分子生物学]