Lehrstuhl für Angewandte Physik and Center for NanoScience, Ludwig-Maximilians-Universität München, Amalienstrasse 54, 80799, Munich, Germany.
Center for Integrated Protein Science Munich, (CIPSM), Ludwig-Maximilians-Universität München, Butenandtstrasse 5-13, 81377, Munich, Germany.
Angew Chem Int Ed Engl. 2018 Sep 24;57(39):12666-12669. doi: 10.1002/anie.201805034. Epub 2018 Aug 31.
Covalent surface immobilization of proteins for binding assays is typically performed non-specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4'-phosphopantetheinyl transferase, sortase A, and asparaginyl endopeptidase) for biolayer interferometry surface modification. All of these enzymes can be used to site-specifically and covalently ligate proteins of interest via short recognition sequences. The enzymes function under mild conditions and thus immobilization does not affect the receptors' functionality. We successfully employed this enzymatic surface functionalization approach to study the binding kinetics of two different receptor-ligand pairs.
共价表面固定化蛋白质用于结合分析通常通过赖氨酸残基非特异性地进行。然而，那些在结合口袋附近有赖氨酸的受体，或者在传感器表面存在时受到静电排斥的受体，可能很难探测。为了克服这些限制并提高表面功能化的均一性，我们对三种不同的酶偶联策略（4'-磷酸泛酰巯基乙胺转移酶、Sortase A 和天冬酰胺内肽酶）进行了适应性优化，用于生物层干涉表面修饰。所有这些酶都可以通过短的识别序列，特异性和共价连接感兴趣的蛋白质。这些酶在温和的条件下发挥作用，因此固定化不会影响受体的功能。我们成功地将这种酶表面功能化方法应用于研究两种不同的受体-配体对的结合动力学。
