Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, School of Medicine, University of Leeds, Leeds LS2 9JT, UK.
Cell Chem Biol. 2022 Feb 17;29(2):339-350.e10. doi: 10.1016/j.chembiol.2021.07.005. Epub 2021 Jul 28.
There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.
有许多有效的方法可以将蛋白质连接到末端。然而，由于较低的灵活性和可变的环境，连接在环上是困难的。在这里，我们开发了 DogCatcher，这是一种与 DogTag 肽自发形成异肽键的蛋白质。最初，DogTag/DogCatcher 是通过分裂肺炎链球菌黏附素产生的。我们通过合理设计和进化优化了 DogTag/DogCatcher，将反应速率提高了 250 倍，并建立了 DogCatcher 的毫摩尔溶解度。当融合到蛋白质末端时，DogTag/DogCatcher 的反应速度比 SpyTag003/SpyCatcher003 慢。然而，插入荧光蛋白或酶的环中时，DogTag 的反应速度比 SpyTag003 快得多。像许多膜蛋白一样，离子通道 TRPC5 没有暴露在表面的末端。TRPC5 细胞外环中的 DogTag 允许在 1 分钟内正常钙流和细胞特异性共价标记。DogTag/DogCatcher 在各种条件下反应，浓度为纳摩尔，转化率为 98%。对环友好的连接应该扩展创建蛋白质结构的工具包。
