Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization

Chemistry - A European Journal, vol. 26, pp. 13568–13572

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.

Authors 5

1. Zhi Zou Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI–Leibniz-Institute for Interactive Materials Forckenbeckstraβe 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

2. Diana M. Maté Aachen

   DWI – Leibniz Institute for Interactive Materials · Centro de Biología Molecular Severo Ochoa · Universidad Autónoma de Madrid

   Affiliation as printed

   Current address: Center of Molecular Biology “Severo Ochoa” Universidad Autónoma de Madrid Nicolás Cabrera 1 28049 Madrid Spain

   DWI–Leibniz-Institute for Interactive Materials Forckenbeckstraβe 50 52074 Aachen Germany

3. Maximilian Nöth Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI–Leibniz-Institute for Interactive Materials Forckenbeckstraβe 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

4. Felix Jakob Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI–Leibniz-Institute for Interactive Materials Forckenbeckstraβe 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany

5. Ulrich Schwaneberg corresponding Aachen Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI–Leibniz-Institute for Interactive Materials Forckenbeckstraβe 50 52074 Aachen Germany

   Institute of Biotechnology RWTH Aachen University Worringerweg 3 52074 Aachen Germany

   Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany