在酵母中高效地产生酸甘氨酸α-氧化单氧酶,用于蛋白质C端功能化
Tong Zhu1, Xuanshuo Zhang2, Ruifeng Li1
1AIM center, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
International journal of biological macromolecules
|February 28, 2024
概括
我们开发了一种高效的方法,用于在酵母中产生酸甘氨酸α-氧化单氧酶 (PHM),从而使改性生物活性的可扩展制造成为可能. 这一突破简化了生产,并增强了制药应用的潜力.
科学领域:
- 生物化学 生物化学
- 蛋白质工程是指蛋白质工程.
- 生物技术是生物技术.
背景情况:
- 基甘氨酸α-基化单氧酶 (PHM) 对于生物活性的C终端化至关重要.
- 现有的PHM生产方法面临动物细胞培养复杂性和细菌宿主局限性 (缺乏糖化) 的挑战.
研究的目的:
- 建立一个高效和可扩展的复合生产系统,用于Caenorhabditis elegans PHM (CePHM).
- 调查N-糖化对CePHM活性的影响.
- 为了证明重组CePHM在制药生产中的实用性.
主要方法:
- 在Pichia pastoris酵母中对CePHM的重组表达.
- 质谱测量用于识别N-糖化位.
- 生物化学测试以评估酶活性.
- 作为模型基质的乌比奎的C端化.
主要成果:
- 实现了复合CePHM的高时空产量28.8U/L/天.
- 在N182残留物中确定了N-糖化,影响了酶活性.
- 成功地在30g/L的基质负载下进行了乌比奎的C端化,适合制药制造.
结论:
- 皮奇亚牧草是高产CePHM生产的有效宿主.
- 再组合的CePHM表现出受N-糖基化影响的活性.
- 这种方法为可扩展的C-终端修饰生物活性的制造提供了一个有希望的平台.
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