Kurpiers Thomas, Mootz Henning D
Technische Universität Dortmund, Fakultät Chemie-Chemische Biologie, Otto-Hahn-Strasse 6, 44229 Dortmund, Germany.
Chembiochem. 2008 Sep 22;9(14):2317-25. doi: 10.1002/cbic.200800319.
The selective modification of proteins with a synthetic probe is of central interest for many aspects of protein chemistry. We have recently reported a new approach in which a short cysteine-containing tag (CysTag) fused to one part of a split intein is first modified with a sulfhydryl-reactive probe. In a second step, protein trans-splicing is used to link the labelled CysTag to a target protein that has been expressed in fusion with the complementary split intein fragment. Here, we present the generation and biochemical characterisation of the artificially split Mycobacterium xenopi GyrA intein. We show that this split intein is active without a renaturation step and that it provides a significant improvement for the CysTag protein-labelling approach in terms of product yields and target protein tolerance. Two proteins with multiple cysteine residues, human growth hormone and a multidomain nonribosomal peptide synthetase, were site-specifically modified with high yields. Our approach combines the benefits of the plethora of commercially available cysteine-reactive probes with a straightforward route for their site-specific incorporation even into complex and cysteine-rich proteins.
用合成探针选择性修饰蛋白质是蛋白质化学诸多方面的核心研究内容。我们最近报道了一种新方法，即首先用巯基反应性探针修饰与分裂内含肽的一部分融合的含半胱氨酸短标签（CysTag）。第二步，利用蛋白质反式剪接将标记的CysTag与已与互补分裂内含肽片段融合表达的靶蛋白连接起来。在此，我们展示了人工分裂的嗜肺分枝杆菌GyrA内含肽的构建及其生化特性。我们表明，这种分裂内含肽无需复性步骤即可激活，并且在产物产量和靶蛋白耐受性方面为CysTag蛋白标记方法带来了显著改进。两种含有多个半胱氨酸残基的蛋白质，即人生长激素和一种多结构域非核糖体肽合成酶，被高产率地进行了位点特异性修饰。我们的方法结合了大量市售半胱氨酸反应性探针的优点，以及将它们位点特异性掺入甚至是复杂且富含半胱氨酸的蛋白质中的直接途径。
