Merten Hannes, Schaefer Jonas V, Brandl Fabian, Zangemeister-Wittke Uwe, Plückthun Andreas
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Institute of Pharmacology, University of Bern, Bern, Switzerland.
Methods Mol Biol. 2019;2033:253-273. doi: 10.1007/978-1-4939-9654-4_17.
For biomedical applications, proteins may require conjugation to small and large molecules. Typical examples are dyes for imaging, cytotoxic effector molecules for cell killing, or half-life extension modules for optimized pharmacokinetics. Although many conjugation strategies are straightforward to apply, most of them do not enable site-specific and orthogonal conjugation, and do not yield a defined stoichiometry. Moreover, techniques offering these desirable features often rely on complex expression procedures and suffer from low production yields. A more promising manufacturing strategy for flexible, site-specific and stoichiometrically defined payloading of proteins is the combination of click chemistry and thiol-maleimide conjugation, which even enables dual labeling when used consecutively. Here, we describe as an example the production of Designed Ankyrin Repeat Proteins (DARPins), a non-IgG binding scaffold, in a specific E. coli strain to obtain high yields of protein carrying both a thiol and an azide group. We provide straightforward protocols for strain-promoted azide-alkyne cycloaddition (SPAAC) and thiol-maleimide conjugation, and furthermore compare these conjugation chemistries with existing alternatives like copper-catalyzed azide-alkyne cycloaddition (CuAAC). Finally, detailed instructions for reactivity analysis and yield estimations of the reactions are provided.
对于生物医学应用而言，蛋白质可能需要与小分子和大分子进行缀合。典型的例子包括用于成像的染料、用于细胞杀伤的细胞毒性效应分子，或用于优化药代动力学的半衰期延长模块。尽管许多缀合策略易于应用，但大多数都无法实现位点特异性和正交缀合，也无法产生确定的化学计量比。此外，具备这些理想特性的技术通常依赖复杂的表达程序，且产量较低。一种更有前景的用于蛋白质灵活、位点特异性和化学计量定义负载的制造策略是将点击化学与硫醇-马来酰亚胺缀合相结合，连续使用时甚至能实现双重标记。在此，我们以在特定大肠杆菌菌株中生产设计锚蛋白重复序列蛋白（DARPins，一种非免疫球蛋白结合支架）为例，以获得高产率的同时携带硫醇和叠氮基团的蛋白质。我们提供了用于应变促进的叠氮化物-炔烃环加成（SPAAC）和硫醇-马来酰亚胺缀合的直接方案，此外还将这些缀合化学方法与铜催化的叠氮化物-炔烃环加成（CuAAC）等现有替代方法进行了比较。最后，提供了反应活性分析和产率估算的详细说明。
