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拟南芥Alpha-dioxygenase1的原核表达、纯化及活性的检测

DOI: 10.7668/hbnxb.2008.03.003, PP. 9-12

Keywords: Alpha-dioxygenase1,重组表达,2,4-DNP,亲和纯化,活性

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Abstract:

从经水杨酸处理的拟南芥开花期植株中获得cDNA,扩增得到Alpha-dioxygenase1(DOX1)基因,进行原核表达、纯化和生物活性检测。利用原核表达载体pMAL-c4x在T7ExpressCompetentE.coli和BL21(DE3)-RIPLcodon+菌株中表达DOX1,经AmyloseResin亲和层析柱纯化。SDS-PAGE结果表明,重组融合蛋白在BL21(DE3)-RIPLcodon+中的表达通过灰度值比较分析以可溶性为主,表达率约3.7%,纯化的DOX1纯度可达45%。愈创木酚法表明,可溶性重组蛋白不具有过氧化物酶活性;2,4-DNP法测试表明可溶性重组蛋白具有脂肪酸双加氧酶活性。

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