Liebscher Sandra, Bordusa Frank
Charles-Tanford-Protein Center, Institute of Biochemistry/Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle, Germany.
Methods Mol Biol. 2019;2033:95-115. doi: 10.1007/978-1-4939-9654-4_8.
Site-specific incorporation of artificial functionalities into protein targets is an important tool in both basic and applied research and can be a major challenge to protein chemists. Chemical labeling methods often targeting multiple positions within a protein and therefore suffer from lack of specificity. Enzymatic protein modification is an attractive alternative due to the inherent regioselectivity and stereoselectivity of enzymes. In this contribution we describe the application of the highly specific trypsin variant named trypsiligase for the site-specific modification of virtual any target protein. We present two general routes of modification resulting in either N- or C-terminal functionalized protein products. Both reaction regimes proceed under mild and bioorthogonal conditions in a short period of time which result in homogeneously modified proteins bearing the artificial functionality exclusively at the desired position. We detail protocols for the expression and purification of trypsiligase as well as the construction of peptide or acyl donor ester probes used as substrates for the biocatalyst. In addition, we provide instructions how to perform the ultimate bioconjugation reactions and finally render assistance for the qualitative and quantitative analysis of the reaction course and outcome.
将人工功能基团位点特异性地引入蛋白质靶点是基础研究和应用研究中的一项重要工具，同时也是蛋白质化学家面临的一项重大挑战。化学标记方法通常会靶向蛋白质内的多个位置，因此缺乏特异性。由于酶具有固有的区域选择性和立体选择性，酶促蛋白质修饰是一种有吸引力的替代方法。在本论文中，我们描述了一种名为胰蛋白酶连接酶的高特异性胰蛋白酶变体在几乎任何目标蛋白质的位点特异性修饰中的应用。我们展示了两种通用的修饰途径，可产生N端或C端功能化的蛋白质产物。两种反应体系均在温和且生物正交的条件下短时间内进行，从而得到仅在所需位置带有人工功能基团的均匀修饰蛋白质。我们详细介绍了胰蛋白酶连接酶的表达和纯化方案，以及用作生物催化剂底物的肽或酰基供体酯探针的构建方法。此外，我们提供了如何进行最终生物共轭反应的说明，并最终为反应过程和结果的定性和定量分析提供帮助。
