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pMOX: a new powerful promoter for recombinant protein production in yeast Pichia pastoris
Enzyme and Microbial Technology ( IF 3.4 ) Pub Date : 2020-09-01 , DOI: 10.1016/j.enzmictec.2020.109582
Mohsen Mombeni 1 , Sareh Arjmand 2 , Seyed Omid Ranaei Siadat 2 , Houshang Alizadeh 1 , Alireza Abbasi 1
Affiliation  

AOX1 promoter (pAOX1) is a robust inducible promoter highly preferred for the production of recombinant proteins in Pichia pastoris (P. pastoris). However, repression by other carbon sources and induction by methanol, which is a fire hazard chemical and undesirable for industrial production, are remarkable drawbacks in large-scale use of this promoter. Hence, novel strong regulatory promoters are highly desired. In the present study, the promoter region of methanol oxidase gene (pMOX), from Hansenula polymorpha, was explored for the heterologous expression of foreign proteins in protease deficient and wild type P. pastoris strains. The promoter region of MOX was isolated and replaced with the pAOX1 in the pPINK-HC plasmid. The activity of pMOX and pAOX1 was compared using endoglucanase 3 (CMC3) and endoglucanase II (EgII) enzymes as the reporter proteins. Evaluation of carbon sources on pMOX activity showed complete inactivation in the presence of xylose and sorbitol and high activity by glycerol, glucose and methanol feeding. Furthermore, the results indicated that increasing the gene dosage and using protease deficient-trait significantly increased CMC3 and EgII expression under the control of pMOX. In conclusion, in this study, a new small powerful and methanol-free promoter is introduced for recombinant protein production in yeast P. pastoris.

中文翻译:

pMOX:一种用于酵母毕赤酵母中重组蛋白生产的强大启动子

AOX1 启动子 (pAOX1) 是一种强大的诱导型启动子,非常适合在毕赤酵母 (P. pastoris) 中生产重组蛋白。然而,其他碳源的抑制和甲醇的诱导,这是一种火灾危险化学品,不利于工业生产,是大规模使用这种促进剂的显着缺点。因此,非常需要新的强调节启动子。在本研究中,探讨了来自多形汉逊酵母的甲醇氧化酶基因 (pMOX) 的启动子区域在蛋白酶缺陷型和野生型毕赤酵母菌株中异源蛋白质的异源表达。分离出 MOX 的启动子区域,并用 pPINK-HC 质粒中的 pAOX1 替换。使用内切葡聚糖酶 3 (CMC3) 和内切葡聚糖酶 II (EgII) 作为报告蛋白比较 pMOX 和 pAOX1 的活性。碳源对 pMOX 活性的评估表明,在木糖和山梨糖醇存在下完全失活,在甘油、葡萄糖和甲醇进料下具有高活性。此外,结果表明,在 pMOX 的控制下,增加基因剂量和使用蛋白酶缺陷性状显着增加了 CMC3 和 EgII 的表达。总之,在本研究中,引入了一种新的小型强力无甲醇启动子,用于在酵母毕赤酵母中生产重组蛋白。结果表明,在 pMOX 的控制下,增加基因剂量和使用蛋白酶缺陷性状显着增加了 CMC3 和 EgII 的表达。总之,在本研究中,引入了一种新的小型强力无甲醇启动子,用于在酵母毕赤酵母中生产重组蛋白。结果表明,在 pMOX 的控制下,增加基因剂量和使用蛋白酶缺陷性状显着增加了 CMC3 和 EgII 的表达。总之,在本研究中,引入了一种新的小型强力无甲醇启动子,用于在酵母毕赤酵母中生产重组蛋白。
更新日期:2020-09-01
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