【精选】5 张金龙-黄曲酶尿酸氧化酶突变体的构建_表达_纯化及生物学活性分析5 张金龙-黄曲酶尿酸氧化酶突变体的构建_表达_纯化及生物学活性分析.pdfVIP

生 物 工 程 学 报 Chin J Biotech 2010, August 25; 26(8): 1102−1107 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2010 CJB, All rights reserved. 医学与免疫生物技术 张金龙，任军，李冰，刘树玲，侯利华，付玲，李建民，陈薇 100071 : 采用融合PCR 的方法将黄曲霉尿酸氧化酶(UOX) 基因的307~309 bp 的TGC(Cys) 突变为GCC(Ala) ，将所获 得的突变体基因克隆到原核表达质粒pET-42a(+) 后转化大肠杆菌BL21(DE3) 。经IPTG 诱导，突变体蛋白 (UOX-Ala 103) 得到高水平的可溶性表达，目的蛋白占总蛋白含量的45 ％。疏水柱及阴离子柱纯化后，UOX-Ala 103 蛋白纯度＞98 ％。 Western blotting 分析证实UOX-Ala 103 能与抗UOX 单抗特异结合。与天然型相比较，其体外生物学活性增加约60 ％， 在高尿酸血症小鼠模型体内也有良好的降解尿酸的活性。 : 黄曲霉尿酸氧化酶，突变体，半胱氨酸，表达，纯化，生物活性 Construction, expression, purification and characterization of mutant of Aspergillus flavus urate oxidase Jinlong Zhang, Jun Ren, Bing Li, Shuling Liu, Lihua Hou, Ling Fu, Jianmin Li, and Wei Chen 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 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 vit