球形节杆菌尿酸酶的表达、纯化及其活性
发布时间：2019-07-01 19:06
【摘要】：目的在大肠埃希菌中表达球形节杆菌尿酸酶(uricase,Uri),并进行纯化和检测其活性。方法根据大肠埃希菌遗传密码子偏爱性,结合原核翻译起始序列的局部二级结构自由能最小化原则,优化设计编码Uri蛋白的核苷酸序列,经PCR扩增球形节杆菌uri基因,克隆至载体pET43.1a,构建重组表达质粒pET43.1a-uri,转化感受态大肠埃希菌BL21-odonPlus(DE3)-RIPL,IPTG诱导表达。表达产物经硫酸铵粗纯及DEAE琼脂糖凝胶层析纯化后,经SDS-PAGE分析纯度;参考Uri产品说明书测定蛋白酶活性,并确定其最佳检测温度及pH值。结果重组表达质粒pET43.1a-uri经酶切及测序鉴定构建正确;重组蛋白相对分子质量约33 000,以可溶性形式存在,表达量约占菌体总蛋白的40%;纯化后纯度可达90%以上;酶活性达13.2 U/mg,酶活性检测最佳反应温度为40℃,最佳反应pH值为9.0。结论成功于大肠埃希菌中表达了uri基因,并获得高纯度的Uri蛋白,其酶学性质与天然的球形节杆菌尿酸酶基本一致,为其大规模稳定生产及尿酸酶法检测试剂的配制研究奠定了基础。
[Abstract]:Objective to express uricase (uricase,Uri) in Escherichia coli, purify and detect its activity. Methods according to the genetic codon preference of Escherichia coli and the principle of minimizing the local secondary structure free energy of prokaryotic translation initiation sequence, the nucleotides encoding Uri protein were optimized. The uri gene of Bacillus globularis was amplified by PCR and cloned into vector pET43.1a, to construct the recombinant expression plasmid pET43.1a-uri, to transform the receptive Escherichia coli BL21-odonPlus (DE3)-RIPL,IPTG to induce expression. After purification by ammonium sulfate and DEAE agarose gel chromatography, the purity of the expressed product was analyzed by SDS-PAGE, and the protease activity was determined with reference to Uri product specification, and the optimum detection temperature and pH value were determined. Results the recombinant expression plasmid pET43.1a-uri was constructed correctly by restriction enzyme digestion and sequencing, the relative molecular weight of the recombinant protein was about 33000, which existed in soluble form, accounting for 40% of the total bacterial protein, the purity was over 90%, the enzyme activity was 13.2 U 鈮，
本文编号：2508725
[Abstract]:Objective to express uricase (uricase,Uri) in Escherichia coli, purify and detect its activity. Methods according to the genetic codon preference of Escherichia coli and the principle of minimizing the local secondary structure free energy of prokaryotic translation initiation sequence, the nucleotides encoding Uri protein were optimized. The uri gene of Bacillus globularis was amplified by PCR and cloned into vector pET43.1a, to construct the recombinant expression plasmid pET43.1a-uri, to transform the receptive Escherichia coli BL21-odonPlus (DE3)-RIPL,IPTG to induce expression. After purification by ammonium sulfate and DEAE agarose gel chromatography, the purity of the expressed product was analyzed by SDS-PAGE, and the protease activity was determined with reference to Uri product specification, and the optimum detection temperature and pH value were determined. Results the recombinant expression plasmid pET43.1a-uri was constructed correctly by restriction enzyme digestion and sequencing, the relative molecular weight of the recombinant protein was about 33000, which existed in soluble form, accounting for 40% of the total bacterial protein, the purity was over 90%, the enzyme activity was 13.2 U 鈮，
本文编号：2508725
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