Abstract

SNAP-tag ^® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach, by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP-tag ^® and its relative thermostable version (SsOGT-H⁵ ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag.

 抽象的

SNAP- tag ^®是一种使用苄基鸟嘌呤 (BG) 衍生物作为底物来标记蛋白质/酶的强大技术。尽管可商业购买或临时生产，但它们的合成和纯化是必要的，从而增加了时间和成本。为了解决这一限制，我们在这里建议对此方法进行修订，通过执行化学酶方法，使用含有叠氮基团的 BG 底物，该叠氮基团与苯甲基环之间有适当距离的间隔基。事实证明^， SNAP- tag®及其相对耐热版本 ( Ss OGT- H ⁵ ) 在此基材上非常活跃。这些标签在酶促反应中的稳定性使得与催化半胱氨酸连接的叠氮部分暴露于溶剂成为可能，适合随后通过叠氮-炔惠斯根环加成与 DBCO 衍生物缀合。我们的研究建议加强和改进这种自标记蛋白质标签的生物技术应用。