尿酸氧化酶的表达纯化及活性形式鉴定

Expression and purification of urate oxidase and identification of its bioactive type

目的:在大肠杆菌内表达具有活性的黄曲霉尿酸氧化酶。方法:用重叠PCR从黄曲霉(Aspergillusflavus)3.1398中钓取了尿酸氧化酶(urateoxidase,UO,EC1.7.3.3)基因,克隆到原核表达载体pET22b中,转化大肠杆菌BL21(DE3),筛选到高表达的重组转化子菌株。经乳糖诱导目的蛋白表达,阴离子交换柱纯化,获得纯品。SDS PAGE分析样品纯度。用尿酸缓冲液测定酶活性,用戊二醛交联结合质谱法测定相对分子质量。结果:目的蛋白为胞内可溶性表达,表达量达菌体总蛋白的35%,纯化后的样品纯度达99%,其酶比活力可达35U/mg。经质谱测定重组UO单体的相对分子质量为34×103。交联后相对分子质量为160×103。结论:黄曲霉尿酸氧化酶可在大肠杆菌中可溶表达,并具有活性,其活性形式为四聚体。

Objective:To study the expression in active form of the urate oxidase gene of Aspergillus flavus in Escherichia coli. Methods: A urate oxidase(uricase,EC1.7.3.3)gene from A.flavus 3.1398 was cloned by overlapping PCR amplification with primers derived from conserved regions of published uricase DNA sequence.The cloned gene was inserted into prokaryote expression vector pET22b after digestion with NdeⅠ and EcoRⅠ, then created the recombinant plasmid pET-UO.It transformed the expression host bacterium E.coli BL21(DE3) competent cells that expressed recombinant soluble proteins with the induction of lactose. The recombinant urate oxidase was purified through anion-exchange chromatography. Its activity was measured with the substrate uric acid. Its molecular weight was determined by MALDI-TOF mass spectrometry after cross-linking with glutaraldehyde. SDS-PAGE was used to analyze the purity of the purified protein. Results: The expression level of the soluble urate oxidase reached to about 35% of the total proteins of the cell.After purification the purity of uricase approached 99%. The enzymatic activity of uricase was 35 U/mg. The protein molecular weight was 34×10 3in monomer form and 160×10 3 after cross-linking. Conclusion: The urate oxidase from A. flavus expressed in E. coli is an active tetramer.