Kuwahara Kana, Harada Kazuki, Yamagoshi Ryohei, Yamamoto Takenori, Shinohara Yasuo
Institute for Genome Research, University of Tokushima, Kuramoto-cho-3, Tokushima, 770-8503, Japan.
Mol Cell Biochem. 2015 Jun;404(1-2):25-30. doi: 10.1007/s11010-015-2362-x. Epub 2015 Feb 20.
The characteristics of antibody delivery into cultured HeLa cells were examined using two delivery systems. Both systems used a cell-penetrating peptide as a tool for intrusion of an antibody into the cells, but either a "protein A derivative" or "hydrophobic motif" was employed to capture the antibody. When we examined the uptake of the Alexa Fluor-labeled antibody by the use of these two systems, both systems were found to effectively deliver the antibody into the cultured cells. However, when we compared the amount of antibody delivered by these systems with the amount of transferrin uptake, the former was 10 times smaller than the latter. The lower efficiency of antibody delivery than transferrin uptake seemed to be attributable to the involvement of the antibody delivery reagent, which failed to catch the antibody molecule. This interpretation was validated by an experiment using a larger amount of antibody, and the amount of antibody delivered by the "protein A derivative" system under this condition was determined to be 13 ng proteins/10(5) cells. The antibody delivery achieved by the "protein A derivative" or "hydrophobic motif" showed two differences, i.e., a difference in intracellular distribution of the delivered antibody molecules and a difference in the fluorescence spectrum observed with cellular lysates. Possible reasons for these differences between the two delivery systems are discussed.
使用两种递送系统研究了抗体递送至培养的HeLa细胞中的特性。两种系统均使用细胞穿透肽作为抗体侵入细胞的工具，但分别采用“蛋白A衍生物”或“疏水基序”来捕获抗体。当我们使用这两种系统检测Alexa Fluor标记抗体的摄取情况时，发现两种系统均能有效地将抗体递送至培养细胞中。然而，当我们将这些系统递送的抗体量与转铁蛋白摄取量进行比较时，前者比后者小10倍。抗体递送效率低于转铁蛋白摄取似乎归因于抗体递送试剂，其未能捕获抗体分子。使用大量抗体进行的实验验证了这一解释，在此条件下“蛋白A衍生物”系统递送的抗体量被确定为13 ng蛋白质/10⁵个细胞。“蛋白A衍生物”或“疏水基序”实现的抗体递送表现出两个差异，即递送的抗体分子在细胞内分布的差异以及细胞裂解物观察到的荧光光谱的差异。讨论了这两种递送系统之间这些差异的可能原因。
