Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris
Production and characterization of human extracellular superoxide dismutase in the methylotrophic yeast Pichia pastoris
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DOI：
10.1021/jf061379x
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发表时间：
2006-10-18
影响因子：
6.1
通讯作者：
Chen, Chuan-Mu
中科院分区：
文献类型：
--
作者：
Chen, Hsiao-Ling;Yen, Chih-Ching;Chen, Chuan-Mu
活性氧与各种疾病相关，包括心血管疾病、神经系统疾病和肺部疾病。细胞外超氧化物歧化酶（ECSOD）是一种由细胞分泌的抗氧化酶，可防止活性氧的过度产生。我们表达ECSOD基因分离的人主动脉平滑肌cDNA文库中的甲醇营养型酵母巴斯德毕赤酵母。用醇氧化酶1基因（AOX 1）的诱导型启动子和酵母α-交配因子信号肽构建了合成分泌盒。在诱导条件下生长的一些转化体中，ECSOD占总蛋白的25%。甲醇诱导36 h后，ECSOD以约440 mg/L的浓度输出到培养基中，抗氧化活性为760 +/- 20 U/mg ECSOD。转化后的酵母细胞对热休克和H2 O2氧化应激的耐受性增强，表明毕赤酵母表达的人ECSOD具有多种生物学功能。我们的数据表明，甲基营养型酵母诱导系统是适合于大规模生产的酶活性的人ECSOD。
Reactive oxygen species are associated with various diseases including cardiovascular diseases, neurological disorders, and pulmonary diseases. Extracellular superoxide dismutase (ECSOD) is an antioxidant enzyme secreted by cells to prevent overproduction of reactive oxygen species. We expressed an ECSOD gene isolated from a human aortic smooth muscle cDNA library in the methylotrophic yeast Pichia pastoris. A synthetic secretion cassette was constructed with the inducible promoter of the alcohol oxidase 1 gene (AOX1) and the yeast alpha-mating factor signal peptide. As much as 25% of the total protein was ECSOD in some transformants grown under inducing conditions. After 36 h of methanol induction, ECSOD was exported into the culture medium at a concentration of approximately 440 mg/L with an antioxidative activity of 760 +/- 20 U/mg ECSOD. Transformed yeast cells were more resistant to heat shock and H2O2 oxidative stress, indicating that the human ECSOD expressed by P. pastoris had multiple biological functions. Our data suggest that the methylotrophic yeast inducible system is suitable for large-scale production of enzymatically active human ECSOD.