Tirat Aline, Freuler Felix, Stettler Thomas, Mayr Lorenz M, Leder Lukas
Novartis Institutes for Biomedical Research, Discovery Technologies, CH-4056 Basel, Switzerland.
Int J Biol Macromol. 2006 Aug 15;39(1-3):66-76. doi: 10.1016/j.ijbiomac.2006.01.012. Epub 2006 Feb 28.
Modern drug discovery strongly depends on the availability of target proteins in sufficient amounts and with desired properties. For some applications, proteins have to be produced with specific modifications such as tags for protein purification, fluorescent or radiometric labels for detection, glycosylation and phosphorylation for biological activity, and many more. It is well known that covalent modifications can have adverse effects on the biological activity of some target proteins. It is therefore one of the major challenges in protein chemistry to generate covalent modifications without affecting the biological activity of the target protein. Current procedures for modification mostly rely on non-specific labelling of lysine or cysteine residues on the protein of interest, but alternative approaches dedicated to site-specific protein modification are being developed and might replace most of the commonly used methodologies. In this study, we investigated two novel methods where target proteins can be expressed in E. coli with a fusion partner that allows protein modification in a covalent and highly selective manner. Firstly, we explored a method based on the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (hAGT) as a fusion tag for site-directed attachment of small molecules. The AGT-tag (SNAP-tag) can accept almost any chemical moiety when it is attached to the guanine base through a benzyl group. In our experiments we were able to label a target protein fused to the AGT-tag with various fluorophores coupled to O6-benzylguanine. Secondly, we tested in vivo and in vitro site-directed biotinylation with two different tags, consisting of either 15 (AviTag) or 72 amino acids (BioEase tag), which serve as a substrate for bacterial biotin ligase birA. When birA protein was co-expressed in E. coli biotin was incorporated almost completely into a model protein which carried these recognition tags at its C-terminus. The same findings were also obtained with in vitro biotinylation assays using pure birA independently over-expressed in E. coli and added to the biotinylation reaction in the test tube. For both biotinylation methods, peptide mapping and LC-MS proved the highly site-specific modification of the corresponding tags. Our results indicate that these novel site-specific labelling reactions work in a highly efficient manner, allow almost quantitative labelling of the target proteins, have no deleterious effect on the biological activity and are easy to perform in standard laboratories.
现代药物发现强烈依赖于具有足够数量和所需特性的靶蛋白。对于某些应用，蛋白质必须进行特定修饰，例如用于蛋白质纯化的标签、用于检测的荧光或放射性标记、用于生物活性的糖基化和磷酸化等等。众所周知，共价修饰可能会对某些靶蛋白的生物活性产生不利影响。因此，在不影响靶蛋白生物活性的情况下进行共价修饰是蛋白质化学中的主要挑战之一。目前的修饰方法大多依赖于对目标蛋白上赖氨酸或半胱氨酸残基的非特异性标记，但致力于位点特异性蛋白质修饰的替代方法正在开发中，可能会取代大多数常用方法。在本研究中，我们研究了两种新方法，通过融合伴侣在大肠杆菌中表达靶蛋白，该融合伴侣允许以共价和高度选择性的方式进行蛋白质修饰。首先，我们探索了一种基于人类DNA修复蛋白O6-烷基鸟嘌呤-DNA烷基转移酶（hAGT）作为小分子定点连接融合标签的方法。当AGT标签（SNAP标签）通过苄基连接到鸟嘌呤碱基时，它几乎可以接受任何化学基团。在我们的实验中，我们能够用与O6-苄基鸟嘌呤偶联的各种荧光团标记与AGT标签融合的靶蛋白。其次，我们用两种不同的标签在体内和体外进行了定点生物素化测试，这两种标签分别由15个氨基酸（Avi标签）或72个氨基酸（BioEase标签）组成，它们作为细菌生物素连接酶birA的底物。当birA蛋白在大肠杆菌中共表达时，生物素几乎完全掺入到在其C末端带有这些识别标签的模型蛋白中。使用在大肠杆菌中独立过表达并添加到试管中的生物素化反应中的纯birA进行体外生物素化测定，也得到了相同的结果。对于这两种生物素化方法，肽图谱分析和液相色谱-质谱证明了相应标签的高度位点特异性修饰。我们的结果表明，这些新的位点特异性标记反应高效运行，几乎可以对靶蛋白进行定量标记，对生物活性没有有害影响，并且易于在标准实验室中进行。
