Abstract

Cancer treatments continue to have many disadvantages. Reactive oxygen species, such as H₂O₂, in high concentrations, can cause cytotoxicity to cells, being even greater in cancer cells. One of the H₂O₂-producing enzymes is glucose oxidase; its application in cancer treatment should be explored. In this work, the extracellular expression of the mutated recombinant enzyme glucose oxidase was carried out in the eukaryotic expression system Pichia pastoris SMD1168, through the modification and optimization of the gox gene of Aspergillus niger to improve its expression in yeast and its purification. Also, the secretion signal of the alpha-mating factor from Saccharomyces cerevisiae was added to the gene for extracellular expression, and it was inserted into the expression vector pPIC3.5k. The extracellular expression of the enzyme facilitated purification by anion exchange chromatography; the purification was corroborated by SDS-PAGE, with a molecular weight of its subunit between 63 kDa and 100 kDa. The mutated recombinant enzyme glucose oxidase showed greater anticancer activity compared to the commercial glucose oxidase and could have potential for cancer treatment.

Key points

• Pichia pastoris is an excellent eukaryotic expression system for proteins that need post-translational modifications.

• Extracellular expression facilitates protein purification.

• Glucose oxidase has potential application in cancer treatment.

中文翻译：

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酵母表达的重组葡萄糖氧化酶相对于商业酶的体外抗癌活性增强

摘要

癌症治疗继续具有许多缺点。高浓度的活性氧（例如H ₂ O _2）会导致对细胞的细胞毒性，在癌细胞中甚至更高。产生H ₂ O _2的酶之一是葡萄糖氧化酶。应探讨其在癌症治疗中的应用。在这项工作中，通过修饰和优化黑曲霉的gox基因以改善其在酵母中的表达和纯化，在真核表达系统毕赤酵母SMD1168中进行了突变的重组酶葡萄糖氧化酶的细胞外表达。另外，来自将酿酒酵母（Saccharomyces cerevisiae）添加到基因中以进行细胞外表达，并将其插入表达载体pPIC3.5k中。酶的细胞外表达促进了阴离子交换色谱法的纯化。通过SDS-PAGE证实纯化，其亚基的分子量在63 kDa和100 kDa之间。与商用葡萄糖氧化酶相比，突变的重组酶葡萄糖氧化酶显示出更高的抗癌活性，并且可能具有治疗癌症的潜力。

关键点

•巴斯德毕赤酵母是需要翻译后修饰的蛋白质的极佳真核表达系统。

•细胞外表达促进蛋白质纯化。

•葡萄糖氧化酶在癌症治疗中具有潜在的应用。