[黄曲霉尿酸氧化酶突变体的构建、表达、纯化及鉴定]

[Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase].

作者信息

Zhang Jinlong, Ren Jun, Li Bing, Liu Shuling, Hou Lihua, Fu Ling, Li Jianmin, Chen Wei

机构信息

State Key Laboratory of Pathogens and Biosecurity, Laboratory of Applied Molecular Biology, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100071, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2010 Aug;26(8):1102-7.

PMID:21090115

Abstract

We converted the TGC codon (307-309 bp) of Aspergillus flavus urate oxidase (UOX) gene to a GCC codon by using fusion PCR techniques to produce a C103A mutant. This gene was cloned into expression vector pET-42a (+) and then transformed into Escherichia coli BL21 (DE3). The mutant protein (UOX-Ala103) was expressed in soluble form at high levels after induction with IPTG The expressed rUOX-Ala103 accounted for about 45% of total bacterial proteins, rUOX-Ala103 of up to 98% purity was obtained after purified using hydrophobic interaction and anion exchange. Western blotting showed that the anti-UOX antibody specifically recognized rUOX-Ala103. The mutant protein showed a 60% increased in vitro biological activities compared with native protein, and performed a good activity of degrading the uric acid in vivo.

摘要

我们通过融合PCR技术将黄曲霉尿酸氧化酶（UOX）基因的TGC密码子（307 - 309 bp）转换为GCC密码子，以产生C103A突变体。该基因被克隆到表达载体pET - 42a（+）中，然后转化到大肠杆菌BL21（DE3）中。用IPTG诱导后，突变蛋白（UOX - Ala103）以可溶性形式高水平表达。表达的rUOX - Ala103约占细菌总蛋白的45%，经疏水相互作用和阴离子交换纯化后，获得纯度高达98%的rUOX - Ala103。蛋白质免疫印迹表明，抗UOX抗体能特异性识别rUOX - Ala103。与天然蛋白相比，突变蛋白的体外生物学活性提高了60%，并且在体内表现出良好的降解尿酸活性。