Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

Sebastian Kiehstaller; George H. Hutchins; Alessia Amore; Alan Gerber; Mohamed Ibrahim; Sven Hennig; Saskia Neubacher; Tom N. Grossmann

doi:10.1021/acs.bioconjchem.3c00151

Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

Sebastian Kiehstaller, George H. Hutchins, Alessia Amore, Alan Gerber, Mohamed Ibrahim, Sven Hennig, Saskia Neubacher^*, Tom N. Grossmann^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

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.

Original language	English
Pages (from-to)	1114-1121
Number of pages	8
Journal	Bioconjugate chemistry
Volume	34
Issue number	6
Early online date	29 May 2023
DOIs	https://doi.org/10.1021/acs.bioconjchem.3c00151
Publication status	Published - 21 Jun 2023

Bibliographical note

Funding Information:
We thank the European Research Council (ERC proof-of-concept, No. 839088) and the Dutch Research Foundation (NWO, Take-off 18617) for funding. Incircular BV thanks the European Innovation Council for support (EIC transition, No. 101057978).

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

Funding

We thank the European Research Council (ERC proof-of-concept, No. 839088) and the Dutch Research Foundation (NWO, Take-off 18617) for funding. Incircular BV thanks the European Innovation Council for support (EIC transition, No. 101057978).

Funders	Funder number
Dutch Research Foundation
HORIZON EUROPE European Innovation Council	101057978
HORIZON EUROPE European Innovation Council
European Research Council	839088
European Research Council
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	Take-off 18617
Nederlandse Organisatie voor Wetenschappelijk Onderzoek

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title = "Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions",

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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AU - Hennig, Sven

AU - Neubacher, Saskia

AU - Grossmann, Tom N.

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Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

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