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

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

摘要:

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-Ala103) 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-Ala103蛋白纯度＞98％。Western blotting分析证实UOX-Ala103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。

关键词:黄曲霉尿酸氧化酶，突变体，半胱氨酸，表达，纯化，生物活性

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.

Key words:urate oxidase (UOX), mutant, cysteine, expression, purification, biological properties