Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

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Abstract

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.

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APA

Kiehstaller, S., Hutchins, G. H., Amore, A., Gerber, A., Ibrahim, M., Hennig, S., … Grossmann, T. N. (2023). Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions. Bioconjugate Chemistry, 34(6), 1114–1121. https://doi.org/10.1021/acs.bioconjchem.3c00151

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