Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

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 in situ 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.