Department of Biomedical Engineering, Faculty of Engineering, Okayama University of Science, 1-1 Ridaicho, Okayama 700-0005, Japan.
Bioconjug Chem. 2012 Oct 17;23(10):2025-31. doi: 10.1021/bc300030d. Epub 2012 Sep 25.
Protein cationization techniques are powerful protein transduction methods for mammalian cells. As we demonstrated previously, cationized proteins with limited conjugation to polyethylenimine have excellent ability to enter into cells by adsorption-mediated endocytosis [Futami, J., et al. (2005) J. Biosci. Bioeng. 99, 95-103]. In this study, we show that proteins with extensive and uniform cationization covering the protein surface reach the cytoplasm and nucleus more effectively than proteins with limited cationic polymers or proteins that are fused to cationic peptides. Although extensive modification of carboxylates results in loss of protein function, chicken avidin retains biotin-binding ability even after extensive amidation of carboxylates. Using this cationized avidin carrier system, the protein transduction ability of variously cationized avidins was investigated using biotinylated protein as a probe. The results revealed that cationized avidins bind rapidly to the cell surface followed by endocytotic uptake. Small amounts of uniformly cationized avidin showed direct penetration into the cytoplasm within a 15 min incubation. This penetration route seemed to be energy dependent and functioned under cellular physiological conditions. A biotinylated exogenous transcription factor protein that penetrated cells was demonstrated to induce target gene expression in living cells.
蛋白质阳离子化技术是一种强大的哺乳动物细胞蛋白转导方法。正如我们之前所证明的，与聚亚乙基亚胺有限结合的阳离子化蛋白通过吸附介导的内吞作用具有极好的进入细胞的能力[Futami，J.等人（2005）J. Biosci. Bioeng. 99，95-103]。在这项研究中，我们表明，与具有有限阳离子聚合物或融合到阳离子肽的蛋白质相比，广泛且均匀地阳离子化覆盖蛋白质表面的蛋白质更有效地进入细胞质和细胞核。尽管广泛修饰羧酸酯会导致蛋白质功能丧失，但鸡亲和素即使在羧酸酯的广泛酰胺化后仍保留生物素结合能力。使用这种阳离子化亲和素载体系统，使用生物素化蛋白作为探针研究了各种阳离子化亲和素的蛋白转导能力。结果表明，阳离子化亲和素迅速与细胞表面结合，随后被内吞摄取。少量均匀阳离子化的亲和素在 15 分钟孵育内直接穿透细胞质。这种穿透途径似乎依赖于能量，并在细胞生理条件下发挥作用。穿透细胞的生物素化外源性转录因子蛋白被证明可在活细胞中诱导靶基因表达。
