Site-specific functionalization of nanobodies: From labeling to cellular uptake

这个合作项目的长期目标是开发和验证新的方法，以可视化和操纵活细胞内的生化过程，并使用荧光标记的蛋白质对细胞摄取进行功能化处理。对于后者，我们将利用循环细胞穿透肽，并对它们进行工程，使其在细胞内切割和释放胞浆和细胞核中的货物蛋白。对于不同靶结构的细胞内结合，我们将使用针对GFP的纳米抗体、DNA复制和修复核心成分PCNA和HIV病毒衣壳蛋白。不同的蛋白质定点修饰策略包括内含子表达、琥珀抑制人工氨基酸掺入、新颖的化学修饰策略和生物正交反应将被用于引入荧光标记和人工环状细胞穿透肽等功能模块。正交官能化策略将首先用GFP结合纳米体进行测试。我们将在体外和体内验证修饰的和未修饰的纳米体与它们各自的细胞内靶标的结合。我们将通过活细胞共聚焦显微镜监测和量化修饰纳米体的摄取。对目标结构的生理影响，包括蛋白质相互作用的破坏，将通过自动图像分析进行量化。这项拟议的合作结合了纳米实体工程、生物正交化学和蛋白质半合成、细胞摄取机制、显微镜和细胞生物学方面的专业知识。因此，这种共同努力应该导致开发全新的研究工具，以检测、研究和操纵活细胞中的抗原。

The long-term goal of this collaborative project is to develop and validate novel methods to visualize and manipulate biochemical processes within living cells with fluorescently labeled proteins functionalized for cellular uptake. For the latter, we will utilize cyclic cell penetrating peptides and engineer them for intracellular cleavage and release of the cargo proteins in the cytosol and nucleus of the cell. For intracellular binding of different target structures we will use nanobodies specific against GFP, the DNA replication and repair core component PCNA and the HIV viral capsid protein. Different strategies for site-specific modification of proteins including intein expression, incorporation of artificial amino acids by amber suppression, novel chemoenzymatic modification strategies and bioorthogonal reactions will be employed to introduce functional modules like fluorescent labels and artificial cyclic cell penetrating peptides. Orthogonal functionalization strategies will initially be tested with the GFP binding nanobodies. We will validate the binding of the modified versus unmodified nanobodies in vitro and in vivo to their respective intracellular targets. We will monitor and quantify the uptake of modified nanobodies by live cell confocal microscopy. Physiological effects on target structures including the disruption of protein interactions will be quantified by automated image analysis. This proposed collaboration, combines expertise in nanobody engineering, bioorthogonal chemistry and protein semi-synthesis, cellular uptake mechanisms, microscopy and cell biology. Consequently, this joint effort should lead to the development of entirely new research tools to detect, study and manipulate antigens in living cells.