重组人血红素加氧酶2的表达、纯化及性质鉴定

Expression,Purification and Characterization of Recombinant Human Heme Oxygenase 2 in Escherichia coli

血红素加氧酶是广泛存在于哺乳动物细胞中的一类氧化酶,催化血红素氧化生成胆绿素,同时生成三价铁离子和一氧化碳,对调节体内离子平衡,参与机体免疫反应及调节神经系统功能均有重要影响.血红素加氧酶2 （HMOX2）与血红素加氧酶1的功能相似,但分布不同且有着不同的反应动力学,但目前国内外对其研究较少.本文在获得HMOX2全长cDNA的基础上,将其克隆至高表达载体,通过自动诱导系统表达了人血红素加氧酶2重组蛋白（His10-HMOX2）.每升菌液通过Ni-NTA亲和层析和Mono Q阴离子交换层析纯化,最终约可获得92.6 mg纯度为92％蛋白质样品.通过酶活检测,其活性可达到69.51 u/mg,与已报道的血红素加氧酶活性相比较,我们得到的HMOX2具有与之相当的酶活性.同时,通过改变反应温度（20 ～ 95℃）,pH值（pH 6 ～9）等条件,检测了重组His10-HMOX2的催化活性,在温度为40℃和pH为8时,其活性最高.本研究为将来进一步深入了解HMOX2的生化功能及与相关疾病的关系奠定了基础.

Heme oxygenase is an enzyme that catalyzes the degradation of beme,and then produces biliverdin,iron ion,and carbon monoxide.Although heme oxygenase-2 （HMOX2） had similar oxygenase activity as HMOX1,their cellular distribution and reaction kinetics are different.Compared to HMOX1,there are few research focused on HMOX2.In this article,we first cloned human HMOX2 to the pT7 high expression level vector from human cDNA,then we overexpressed HMOX2 by the auto-induction system.The expressed recombinant His10-HMOX2 was purified by Ni-NTA affinity chromatography and Mono Q anion exchange chromatography.The typical yield of recombinant HMOX2 was 92.6 mg from 1 liter culture medium with purity about 92%.The recombinant HMOX2 was assayed for oxygenase activity,showing oxygenase activity of 69.51 u/mg.The oxygenase activity assay also showed that the purified HMOX2 tolerated broad ranges of pH （pH 6 ～ 9） and temperature （20 ～ 95 ℃） The optimum temperature and pH is 40℃ and pH 8.Our research may facilitate further research of HMOX2＇s biochemical activity and its role in the related diseases.