对于容易聚合的口,EGF的高效护送策略与Sparcl1重复
Yuji Kondo1,2, Yuxin Li1, Tetsuya Okajima1,2
1Department of Molecular Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
Molecules (Basel, Switzerland)
|March 13, 2024
概括
研究人员开发了一种新的方法来产生聚合的Notch1 EGF14-15蛋白质,这对抗体发育至关重要. 这种技术增强了O结合N-乙糖胺 (O-GlcNAc) 修饰蛋白质的分泌和净化.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 葡萄糖生物学 葡萄糖生物学
背景情况:
- 皮表皮生长因子 (EGF) 重复是许多蛋白质中发现的结构动机,包括Notch受体.
- 这些重复含有独特的非典型的O链 glycans,如O链 N-乙糖胺 (O-GlcNAc).
- O-GlcNAc 修饰在各种细胞过程中发挥作用,使其研究变得重要.
研究的目的:
- 在小鼠中生成针对O-GlcNAc部分的单克隆抗体Notch1.1.
- 为了克服生产和净化重组Notch1 EGF14-15蛋白质作为免疫原体的挑战.
- 建立一种可扩展的方法来获得O-GlcNAc修饰的Notch1 EGF14-15.
主要方法:
- 在HEK293T细胞中表达复合C端的His6标记的Notch1 EGF14-15.
- Sparcl1的融合作为细胞外护送标签到Notch1 EGF14-15的N端,以增强分泌并防止聚合.
- 使用预分离蛋白酶,有效地从聚变标签中释放Notch1 EGF14-15.
- 产生护送标签的删除突变,以优化细胞外分泌.
主要成果:
- 标准表达方法导致蛋白质聚合和Notch1 EGF14-15的低分泌.
- Sparcl1护送标签促进了融合蛋白的高效细胞外分泌,没有聚合.
- 标签1 EGF14-15被成功净化,并确认在从标签上切割后被O-GlcNAcylated.
- 随行标签长度的优化改善了分泌EGF14-15.5的产量.
结论:
- Sparcl1护送标签策略有效地生产可溶和纯化的O-GlcNAc修饰的Notch1 EGF14-15.
- 这种方法克服了蛋白质聚合的问题,使得从细胞培养超级生物中大规模制备标蛋白质.
- 开发的方法促进了对Notch1.1等蛋白质上特定的糖甘修饰的抗体的产生.
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