A general approach for stabilizing nanobodies for intracellular expression.
A general approach for stabilizing nanobodies for intracellular expression.
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DOI：
10.7554/elife.68253
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发表时间：
2022-11-23
期刊：
影响因子：
7.7
通讯作者：
Cepko CL
中科院分区：
文献类型：
--
作者：
Dingus JG;Tang JCY;Amamoto R;Wallick GK;Cepko CL
常规抗体及其衍生片段由于其体积和结构复杂性而难以针对活细胞中的细胞内靶标展开。纳米抗体提供了一种替代方式，具有细胞内表达的充分记录的实例。尽管它们作为细胞内试剂的前景，但还没有对纳米抗体细胞内表达的系统研究。在这里，我们检查了来自蛋白质数据库的75个纳米抗体的细胞内表达。令人惊讶的是，这些纳米抗体中的大多数在细胞中是不稳定的，通过聚集和清除来说明。使用比较分析和框架诱变，我们开发了一种通用方法，该方法稳定了大多数最初在细胞内不稳定的纳米抗体，而不会显著损害靶结合。这种方法导致鉴定影响测试纳米抗体的细胞内稳定性的不同序列特征。突变稳定的纳米抗体表达被发现延伸到体内环境，在鼠视网膜和E。杆菌这些数据提供了用于细胞内应用的纳米抗体工程的改进，增强了细胞内询问和干预的不断增长的领域。
Conventional antibodies and their derived fragments are difficult to deploy against intracellular targets in live cells, due to their bulk and structural complexity. Nanobodies provide an alternative modality, with well-documented examples of intracellular expression. Despite their promise as intracellular reagents, there has not been a systematic study of nanobody intracellular expression. Here, we examined intracellular expression of 75 nanobodies from the Protein Data Bank. Surprisingly, a majority of these nanobodies were unstable in cells, illustrated by aggregation and clearance. Using comparative analysis and framework mutagenesis, we developed a general approach that stabilized a great majority of nanobodies that were originally unstable intracellularly, without significantly compromising target binding. This approach led to the identification of distinct sequence features that impacted the intracellular stability of tested nanobodies. Mutationally stabilized nanobody expression was found to extend to in vivo contexts, in the murine retina and in E. coli. These data provide for improvements in nanobody engineering for intracellular applications, potentiating a growing field of intracellular interrogation and intervention.