Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt 64287, Germany.
Biomacromolecules. 2024 Aug 12;25(8):5300-5309. doi: 10.1021/acs.biomac.4c00724. Epub 2024 Jul 15.
A strategy for the bioorthogonal immobilization of proteins onto commercially available filter paper is presented. Recently, a two-step approach has been described that relies on covalent immobilization of a linker molecule to paper, followed by enzyme-mediated conjugation of a protein of interest containing an enzyme-recognition tag. Here, this strategy was expanded by evaluating four different chemical and chemoenzymatic reactions and investigating paper loading efficiency and orthogonality. Enhanced green fluorescent protein (EGFP) was used as a model protein to allow quantification of protein loading via fluorescence imaging. Two approaches were identified that showed significantly increased loading efficiencies compared with the previously applied conjugation strategy. Additionally, all four methods were proven orthogonal to each other, allowing simultaneous immobilization of a mixture of proteins to a premodified assembly of two paper sheets.
本文提出了一种将蛋白质生物正交固定在市售滤纸上的策略。最近，已经描述了一种两步法，该方法依赖于将连接分子共价固定在纸上，然后通过酶介导的含有酶识别标签的目标蛋白进行连接。在这里，通过评估四种不同的化学和化学酶反应，并研究纸张负载效率和正交性，扩展了该策略。增强型绿色荧光蛋白（EGFP）被用作模型蛋白，通过荧光成像允许定量蛋白质负载。确定了两种方法，与之前应用的连接策略相比，这两种方法的负载效率显著提高。此外，所有四种方法彼此正交，允许将蛋白质混合物同时固定在预先修饰的两张纸的组装体上。
