Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm
Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm
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DOI：
10.1093/protein/gzq022
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发表时间：
2010-06-01
影响因子：
2.4
通讯作者：
Messer, Anne
中科院分区：
文献类型：
--
作者：
Kvam, Erik;Sierks, Michael R.;Messer, Anne
可溶性抗体片段不仅可作为细胞外靶标的潜在治疗和诊断剂，而且还可作为细胞内功能基因组学、蛋白质组学和基因治疗的“体内抗体”。然而，众所周知，抗体片段在细胞内表达时容易聚集，部分原因是细胞质中不利的氧化还原电位和大分子拥挤。只有一小部分胞内抗体可溶于细胞质，并且对于赋予这种稳定性的序列决定因素知之甚少。通过比较哺乳动物细胞中几种相关的人类单链可变片段和骆驼科动物 V(HH) 的细胞质表达，我们发现体内溶解度很大程度上受 CDR 含量的影响，并且通过细胞质 pH 值的整体负电荷和亲水性的降低而得到改善。我们假设离子排斥和弱疏水相互作用在不同程度上补偿了细胞质中受损的二硫键形成，从而降低了体内聚集的风险。作为原理证明，我们证明，使用高电荷肽标签（3XFLAG 标签，SV40 NLS）通过顺式或反式酸化可适度增强易于聚集的带正电荷的体内可溶性表达。这些发现表明，简单的序列分析和静电操作可能有助于预测和设计来自抗体库的溶解度增强的胞内抗体以供细胞内使用。
Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and weak hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of principle, we demonstrate that the soluble expression of an aggregation-prone positively charged intrabody is modestly enhanced via cis or trans acidification using highly charged peptide tags (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use.