Incircular BV, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Department of Chemistry & Pharmaceutical Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands.
Bioconjug Chem. 2023 Jun 21;34(6):1114-1121. doi: 10.1021/acs.bioconjchem.3c00151. Epub 2023 May 29.
Enzymes are of central importance to many biotechnological and biomedical applications. However, for many potential applications, the required conditions impede enzyme folding and therefore function. The enzyme Sortase A is a transpeptidase that is widely used to perform bioconjugation reactions with peptides and proteins. Thermal and chemical stress impairs Sortase A activity and prevents its application under harsh conditions, thereby limiting the scope for bioconjugation reactions. Here, we report the stabilization of a previously reported, activity-enhanced Sortase A, which suffered from particularly low thermal stability, using the cyclization of proteins (INCYPRO) approach. After introduction of three spatially aligned solvent-exposed cysteines, a triselectrophilic cross-linker was attached. The resulting bicyclic INCYPRO Sortase A demonstrated activity both at elevated temperature and in the presence of chemical denaturants, conditions under which both wild-type Sortase A and the activity-enhanced version are inactive.
酶对于许多生物技术和生物医学应用都具有至关重要的作用。然而，对于许多潜在的应用而言，所需的条件会阻碍酶的折叠，从而影响其功能。Sortase A 是一种转肽酶，被广泛用于进行肽和蛋白质的生物偶联反应。热和化学应激会损害 Sortase A 的活性，并阻止其在恶劣条件下的应用，从而限制了生物偶联反应的范围。在这里，我们报告了使用蛋白质环化（INCYPRO）方法对先前报道的活性增强的 Sortase A 的稳定化，该酶的热稳定性特别低。通过引入三个空间排列的暴露在溶剂中的半胱氨酸，引入了一个三亲电子交联剂。所得的双环 INCYPRO Sortase A 在高温和化学变性剂存在的条件下均具有活性，而野生型 Sortase A 和活性增强型 Sortase A 在这些条件下均无活性。
