Targeting cytoplasmic protein-protein interactions with antibodies remains technically challenging, since antibodies expressed in the cytosol frequently form insoluble aggregates. Existing engineering methods are based on the notion that the estimated net charge at pH 7.4 affects stability; as such, they are unable to overcome this problem. Herein, we report a versatile method for engineering an ultra-stable cytoplasmic antibody (STAND), with a strong estimated net negative charge at pH 6.6, by fusing peptide tags with a highly negative charge and a low isoelectric point. Without the need for complicated amino acid substitutions, we convert aggregation-prone antibodies to STANDs that are useful for inhibiting in vivo transmitter release, modulating animal behaviour, and inhibiting in vivo cancer proliferation driven by mutated Kras-long recognised as an "undruggable" oncogenic protein. The STAND method shows promise for targeting endogenous cytoplasmic proteins in basic biology and for developing future disease treatments.

中文翻译：

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设计用于体内应用的超稳定细胞质抗体。

与抗体靶向细胞质蛋白相互作用的技术仍然具有挑战性，因为在胞质溶胶中表达的抗体经常形成不溶性聚集体。现有的工程方法基于以下概念：pH 7.4时估计的净电荷会影响稳定性；因此，他们无法克服这个问题。本文中，我们报告了一种通过融合具有高负电荷和低等电点的肽标签来工程化超稳定细胞质抗体（STAND），在pH值为6.6时具有强烈的估计净负电荷的通用方法。无需复杂的氨基酸取代，我们将易于聚集的抗体转化为STAND，可用于抑制体内递质释放，调节动物行为，并抑制由突变的Kras-long驱动的体内癌症扩散，这种突变被认为是“不可持久的”致癌蛋白。STAND方法显示出有望在基础生物学中靶向内源性细胞质蛋白并开发未来的疾病治疗方法。