Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics and the Molecular Biology Institute , University of California, Los Angeles , Los Angeles , California 90095 , United States.
Department of Microbiology & Molecular Genetics , University of Texas Health Science Center , Houston , Texas 77030 , United States.
J Am Chem Soc. 2018 Jul 11;140(27):8420-8423. doi: 10.1021/jacs.8b05200. Epub 2018 Jun 28.
Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (SrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created SrtA, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. SrtA mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that SrtA can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.
经过肽和化学物质定点修饰的蛋白质可用作新型治疗药物、成像工具、诊断试剂和材料。然而，目前只有少数酶催化方法可用于选择性地将肽连接到蛋白质的内部位点。在这里，我们发现来自白喉棒状杆菌（Corynebacterium diphtheriae）的菌毛特异性 Sortase 酶（SrtA）可用于通过特定的赖氨酸异肽键将肽连接到蛋白质上。我们通过合理的诱变创建了 SrtA，这是一种高度激活的半胱氨酸转肽酶，可在体外催化异肽键形成。SrtA 介导融合结构域内特定赖氨酸残基的生物偶联，融合结构域来自于棒状杆菌 SpaA 蛋白。肽修饰的产率可超过>95%。我们证明 SrtA 可以与金黄色葡萄球菌的 SrtA 酶一起使用，通过赖氨酸异肽键和主链-肽键实现双重正交蛋白质标记。
