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Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions
Sebastian Kiehstaller1, George H Hutchins2,3, Alessia Amore2,3
1Incircular BV, De Boelelaan 1108, 1081 HZ Amsterdam, The Netherlands.
Bioconjugate Chemistry
|May 29, 2023
Summary
Researchers stabilized Sortase A, an enzyme crucial for bioconjugation, using a novel protein cyclization method. The stabilized enzyme retains function under harsh thermal and chemical conditions, expanding its biotechnological applications.
Area of Science:
- Biochemistry
- Protein Engineering
- Biotechnology
Background:
- Enzymes are vital for biotechnological and biomedical applications.
- Harsh conditions like thermal and chemical stress limit enzyme function, hindering applications.
- Sortase A, a transpeptidase, is widely used for protein and peptide bioconjugation but is sensitive to stress.
Purpose of the Study:
- To enhance the stability of an activity-enhanced Sortase A variant.
- To enable Sortase A function under challenging thermal and chemical conditions.
- To expand the scope of Sortase A-mediated bioconjugation reactions.
Main Methods:
- Employed the in situ protein cyclization (INCYPRO) approach.
- Introduced three spatially aligned, solvent-exposed cysteines into Sortase A.
- Attached a triselectrophilic cross-linker to create a bicyclic enzyme.
Main Results:
- The bicyclic INCYPRO Sortase A exhibited retained activity at elevated temperatures.
- The stabilized enzyme remained active in the presence of chemical denaturants.
- Wild-type and activity-enhanced Sortase A were inactive under these conditions.
Conclusions:
- The INCYPRO approach successfully stabilized Sortase A against thermal and chemical stress.
- Stabilized Sortase A broadens the applicability of bioconjugation under demanding conditions.
- This method offers a pathway for engineering robust enzymes for industrial biotechnology.
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