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

纳米抗体的位点特异性功能化：从标记到细胞摄取

• 批准号: 223438233
• 负责人: Professorin Dr. Maria Cristina Cardoso
• 金额: --
• 依托单位: Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/223438233?language=en
• 关键词: Site; specific; functionalization; nanobodies; labeling

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.

该合作项目的长期目标是开发和验证新方法，以可视化和操纵活细胞内的生化过程，并将荧光标记的蛋白质功能化用于细胞摄取。对于后者，我们将利用环状细胞穿透肽并将其工程化以用于细胞内裂解和释放细胞质和细胞核中的货物蛋白。对于不同靶结构的细胞内结合，我们将使用特异性针对GFP、DNA复制和修复核心组分PCNA和HIV病毒衣壳蛋白的纳米抗体。蛋白质的位点特异性修饰的不同策略，包括内含肽的表达，人工氨基酸的琥珀抑制，新的化学酶修饰策略和生物正交反应的掺入将被用来引入功能模块，如荧光标记和人工环细胞穿透肽。正交功能化策略最初将用GFP结合纳米抗体进行测试。我们将在体外和体内验证修饰的纳米抗体与未修饰的纳米抗体与其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的纳米抗体的摄取。对靶结构的生理效应（包括蛋白质相互作用的破坏）将通过自动图像分析进行定量。这项拟议的合作，结合了纳米抗体工程，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的专业知识。因此，这一共同努力应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。