Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Zhi Zou; Diana M Mate; Maximilian Nöth; Felix Jakob; Ulrich Schwaneberg

doi:10.1002/chem.202002740

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Chemistry. 2020 Oct 27;26(60):13568-13572. doi: 10.1002/chem.202002740. Epub 2020 Aug 18.

Authors

Zhi Zou^{1

2}, Diana M Mate^{2

3}, Maximilian Nöth^{1

2}, Felix Jakob^{1

2}, Ulrich Schwaneberg^{1

2}

Affiliations

¹ Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.
² DWI-Leibniz-Institute for Interactive Materials, Forckenbeckstraβe 50, 52074, Aachen, Germany.
³ Current address: Center of Molecular Biology "Severo Ochoa", Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.

Abstract

Staphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6-fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6-fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Keywords: biocatalysis; protein engineering; site-specificity; sortase A; thermal stability.