DogCatcher allows loop-friendly protein-protein ligation.
DogCatcher allows loop-friendly protein-protein ligation.
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DOI：
10.1016/j.chembiol.2021.07.005
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发表时间：
2022-02-17
影响因子：
8.6
通讯作者：
Howarth M
中科院分区：
文献类型：
--
作者：
Keeble AH;Yadav VK;Ferla MP;Bauer CC;Chuntharpursat-Bon E;Huang J;Bon RS;Howarth M
有许多有效的方法在末端连接蛋白质。然而，由于灵活性较低和环境多变，在环路上连接是困难的。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最初是通过分离肺炎链球菌黏附素产生的。通过合理的设计和进化对DogTag/DogCatcher进行了优化，使反应速度提高了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可在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在不同条件下反应，在纳摩尔浓度下，转化率可达98%。环友好型结扎应该扩展创建蛋白质结构的工具箱。DogCatcher是专为高溶解度和快速反应而设计和繁殖的，在蛋白质环内，DogTag可以比SpyTag003更快地夹紧它的伙伴，通过重新设计致病粘附蛋白，通过DogTag亲环蛋白偶联实现细胞表面离子通道的快速和忠实的荧光标记。许多共轭方法只在暴露的末端起作用。Keeble等人创建了用于高产共价标记的DogTag/DogCatcher。在DogTag环插入后，反应性对条件和维持蛋白质的催化、荧光和离子电导功能具有鲁棒性。
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. Spontaneous transamidation at internal sites harnessing a DogTag/DogCatcher pair DogCatcher is designed and bred for high solubility and rapid reaction Within protein loops DogTag can clamp on its partner faster than SpyTag003 Fast and faithful fluorescent labeling of an ion channel at the cell surface via DogTag Loopophilic protein coupling is achieved here by re-engineering a pathogenic adhesion protein. Many conjugation approaches only function at exposed termini. Keeble et al. create DogTag/DogCatcher for high-yield covalent labeling. Reactivity is robust to conditions and maintains protein function for catalysis, fluorescence and ion conductance after DogTag loop insertion.
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DOI：
10.1002/anie.201411511
发表时间：
2015-03-16
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Akbulut Y;Gaunt HJ;Muraki K;Ludlow MJ;Amer MS;Bruns A;Vasudev NS;Radtke L;Willot M;Hahn S;Seitz T;Ziegler S;Christmann M;Beech DJ;Waldmann H
通讯作者：
Waldmann H
影响因子：
7.3
作者：
Brune KD;Howarth M
通讯作者：
Howarth M
影响因子：
4.1
作者：
通讯作者：
--
影响因子：
4.6
作者：
Bonnet J;Cartannaz J;Tourcier G;Contreras-Martel C;Kleman JP;Morlot C;Vernet T;Di Guilmi AM
通讯作者：
Di Guilmi AM
影响因子：
8
作者：
Cabrita, Lisa D.;Gilis, Dimitri;Bottomley, Stephen P.
通讯作者：
Bottomley, Stephen P.