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Cell adhesion is  an essential aspect of multicellularity. While stable cell interactions usually occur between cells of the same type, transient cell interactions occur between cells of different tissue types, such as between neutrophils and endothelial cells. Selectins are one class of cell adhesion molecules (CAMs) that bind carbohydrate ligands to form transient cell adhesion. They are rod-like proteins with a long extracellular part of variable length ending with the lectin domain,...
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Multiprotein signaling complexes are formed in a dynamic process involving protein-protein interactions at the cytoplasmic domain of transmembrane receptors or enzymatic and non-enzymatic proteins associated with the receptor. These complexes ensure the activation and propagation of intracellular signals that regulate cell functions.
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
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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.

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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.

关键词:
标记1 标记1 标记一个火花.这是一种分泌的分泌.

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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.