当前位置: X-MOL 学术Cell Chem. Bio. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
DogCatcher allows loop-friendly protein-protein ligation
Cell Chemical Biology ( IF 6.6 ) Pub Date : 2021-07-28 , DOI: 10.1016/j.chembiol.2021.07.005
Anthony H Keeble 1 , Vikash K Yadav 1 , Matteo P Ferla 1 , Claudia C Bauer 2 , Eulashini Chuntharpursat-Bon 2 , Jin Huang 1 , Robin S Bon 2 , Mark Howarth 1
Affiliation  

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.



中文翻译:

DogCatcher 允许循环友好的蛋白质-蛋白质连接

有许多有效的方法可以在末端连接蛋白质。但是,由于灵活性较低且环境多变,很难在环路上连接。在这里,我们开发了 DogCatcher,一种与 DogTag 肽形成自发异肽键的蛋白质。DogTag/DogCatcher 最初是通过分裂肺炎链球菌生成的粘附素。我们通过合理的设计和进化优化了 DogTag/DogCatcher,将反应速率提高了 250 倍,并确定了 DogCatcher 的毫摩尔溶解度。当与蛋白质末端融合时,DogTag/DogCatcher 的反应比 SpyTag003/SpyCatcher003 慢。然而,插入荧光蛋白或酶的环中,DogTag 的反应速度比 SpyTag003 快得多。像许多膜蛋白一样,离子通道 TRPC5 没有表面暴露的末端。TRPC5 细胞外环中的 DogTag 允许在 1 分钟内对细胞进行正常的钙通量和特异性共价标记。DogTag/DogCatcher 在不同的条件下以纳摩尔浓度反应,转化率为 98%。循环友好的连接应该扩展用于创建蛋白质结构的工具箱。

更新日期:2021-07-28
down
wechat
bug