Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay
Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay
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DOI：
10.1016/j.bbrc.2015.10.066
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发表时间：
2015-11-27
影响因子：
3.1
通讯作者：
Kim, Yong-Sung
中科院分区：
文献类型：
--
作者：
Kim, Ji-sun;Choi, Dong-Ki;Kim, Yong-Sung
考虑到与许多疾病相关的细胞质蛋白的数量，从活细胞外部开发穿透细胞质的分子是非常需要的。为了在细胞摄取后进入细胞质，细胞穿透分子应该在溶酶体降解之前从中间内体释放出来。然而，将胞质释放的分子池与被困在内吞囊泡中的分子区分开是非常具有挑战性的。在这里，我们描述了一种基于增强的分裂GFP互补系统的方法来直接证明细胞质定位和定量细胞质中穿透细胞质的IgG抗体TMab4。我们产生了与一个GFP片段基因融合的TMab4，并在细胞质中分别建立了表达另一个GFP片段的HeLa细胞，这样，只有当细胞外处理的TMab4在细胞内化后到达细胞质时，才能观察到互补的GFP荧光。利用链亲和素结合肽2和链亲和素之间的高亲和相互作用作为GFP片段各自的融合伙伴，提高GFP互补的敏感性。利用该方法，用1 μ M TMab4对HeLa细胞进行细胞外处理6小时后，估计胞内TMab4的浓度约为170 nM。我们还发现，在细胞内化到活细胞后，近1.3 -43%的内化TMab4分子从胞内囊泡逃逸到胞质中。我们的增强分裂GFP互补试验提供了一种有用的工具，可以直接量化细胞溶胶穿透剂的胞质量，并允许基于细胞的高通量筛选具有增加内体细胞逃逸活性的细胞溶胶穿透剂。(C) 2015爱思唯尔公司版权所有。
Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 mu M TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3 -43% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. (C) 2015 Elsevier Inc. All rights reserved.