Dept of Biochemistry, University of Zurich, Winterthurerstr. 190, 8057 Zurich, Switzerland.
J Control Release. 2015 Feb 28;200:13-22. doi: 10.1016/j.jconrel.2014.12.019. Epub 2014 Dec 17.
Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.
通过蛋白质支架工程和选择技术的进步，可以针对几乎所有目标生成在还原条件下折叠的高度特异性结合蛋白。尽管有巨大的治疗机会，但细胞内应用受到实现细胞质递送的困难的阻碍，甚至正确测量它也很复杂。在这里，我们通过开发基于生物素连接酶的测定法来系统地解决细胞质递送问题，该测定法以通用的方式客观地定量细胞质递送。我们开发了模块化的运输系统，该系统由用于受体靶向的设计的锚蛋白重复蛋白 (DARPin) 和用于细胞内识别的不同 DARPin 以及细菌毒素衍生的成分组成，用于细胞质易位。我们表明，炭疽孔和假单胞菌外毒素 A (ETA) 的易位结构域都能有效地将 DARPin 递送到细胞质中。我们发现，货物的热力学稳定性不能超过炭疽孔的阈值，这可以通过工程来解决，而 ETA 途径似乎没有这种限制。
