猪尿酸氧化酶在大肠杆菌中的表达、纯化与部分酶学性质分析  

Expression in Escherichia coli,purification and enzymatic properties of porcine urate oxidase

作　　者：吴双林[1] 陈斌[2,3] 刘成倩[2] 欧瑜[1] 易建中[2] 

机构地区：[1]中国药科大学生命科学与技术学院,南京210009 [2]上海农业科学院畜牧兽医研究所,上海201106 [3]南京农业大学动物医学院,南京210095

出　　处：《生物工程学报》2009年第11期1664-1670,共7页Chinese Journal of Biotechnology

基　　金：上海市浦江人才计划(No.PJ[20])资助~~

摘　　要：本研究报道了猪尿酸氧化酶(Porcine urate oxidase,pUOX)的原核表达载体的构建、pUOX的蛋白表达条件的优化以及对pUOX经纯化后进行活性检测和酶学性质分析。利用RT-PCR从猪肝总RNA中克隆pUOX,定向插入原核表达载体pET30a(+)中,构建表达载体pET30a(+)/pUOX,并转化到大肠杆菌(Escherichia coli)BL21(DE3)中。重组质粒pET30a(+)/pUOX经双酶切鉴定和序列分析,证实已成功构建了重组表达载体。重组表达菌经IPTG诱导表达了约为41kD的蛋白,与预期分子量一致,并对pUOX蛋白表达条件进行了优化,表达的蛋白主要以包涵体的形式存在于细胞中,包涵体经过变性、复性后,用Ni2+-NTA对复性蛋白进行亲和纯化,并对纯化蛋白进行了活性检测和酶学性质分析,纯化的重组pUOX的比活为50.63IU/mg,并发现重组蛋白在最佳温度、热稳定性等方面与天然pUOX相同,为后续动物实验奠定重要的基础。The aims of this research were to construct prokaryotic expression vector containing the gene of porcine urate oxidase（pUOX）,optimize the conditions of the expression of pUOX in recombinant Escherichia coli BL21（DE3）,and analyze the in vitro activity and the enzymological properties of pUOX.The pUOX gene was amplified by RT-PCR from the extracted total RNA of porcine liver,and was inserted into the prokaryotic expression vector pET30a（＋） to construct a recombinant expression vector pET30a（＋）/pUOX.We identified the recombinant vector by endonuclease digestion and sequence analysis.The pUOX gene was amplified and cloned into the vector pET30a（＋） successfully.And then the recombinant vector was transformed into E.coli BL21（DE3）.The expression of pUOX with a molecular of approximately 41 kD was induced by IPTG.We also optimized the expression conditions of the recombinant protein.The recombinant protein was mostly located in the cytoplasm and it was insoluble.After the inclusion body was solved in 8 mol/L urea and refolding in 2 mol/L urea,the recombinant protein was collected and purified by Ni2＋-NTA column.This recombinant protein had a specific activity of 50.61 IU/mg and showed similar properties of optimum temperature and thermal stability,base on the enzymatic assay and analysis of enzymological properties.These results would help to analyze the in vivo activity by testing animal.

关 键 词：猪尿酸氧化酶 表达 包涵体 纯化 生物活性 

分 类 号：Q78[生物学—分子生物学]