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 thein situcyclization 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.