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人类干扰素α2a (IFNα2a) 在甲基营养酵母Pichia pastoris中的表达和净化

Sofia Chronopoulou¹, Ilias Tsochantaridis¹, Maria Tokamani¹

¹Department of Molecular Biology & Genetics, Democritus University of Thrace, Alexandroupolis, Greece.

Protein expression and purification

|July 19, 2023

概括

研究人员成功地使用Pichia pastoris酵母系统生产了生物活性人类干扰素α2a (IFNα2a). 这为生产这种重要的治疗蛋白质提供了可行的替代方案,用于各种医疗应用.

科学领域:

生物技术是生物技术.
分子生物学分子生物学
蛋白质表达方式

背景情况:

人类干扰素α2a (IFNα2a) 是一种具有抗病毒,抗瘤和免疫抑制功能的关键治疗蛋白.
目前IFNα2a的生产依赖于 prokaryotic 系统 (大肠杆菌),存在潜在的限制.
IFNα2a在临床上用于治疗慢性C型肝炎病毒感染,白血病和某些癌症.

研究的目的:

建立和验证甲基营养酵母Pichia pastoris作为复合人类IFNα2a的表达系统.
在真核系统中实现生物功能IFNα2a的高效表达和分泌.
描述P. pastoris产生的IFNα2a的稳定性和活性.

主要方法:

人类IFNα2acDNA被对P. pastoris进行了编码优化,并被克隆到pPinkα-HC载体中.
使用甲醇诱导表达,并通过PCR测序,SDS-PAGE和Western blot确认分泌.
净化是使用亲和色谱进行的,蛋白质稳定性是通过差分扫描度测量来评估的.
通过测量干扰素刺激基因 (ISG) 的诱导,在A549和HT29细胞中验证了生物活性.

主要成果:

再组合的人类IFNα2a (大约. 19 kDa) 被P. pastoris成功表达和分泌.
净化产生了大量的活性IFNα2a,显示出令人印象深刻的热稳定性.
来自P. pastoris的IFNα2a表现出显著的生物活性,在人类细胞系中诱导ISG.

结论:

皮奇亚牧草的表达系统是一个适合和有效的平台,用于生产生物功能,分泌的人类干扰素α2a.
这项研究为IFNα2a生产提供了一种有价值的替代原生体表达系统.
这些发现支持P. pastoris在大型制造IFNα2a.等治疗性蛋白质方面的潜力.

关键词:

A54949 是一个A549的字体.标签:HT29 标签:HT29 标签:HT29 标签:如果IFNα2a2a 干扰素刺激基因 (ISG) 是指干扰素刺激的基因.这就是Pichia pastoris.

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