Enhancing robustness of sortase A by loop engineering and backbone cyclization

10/08/2020
  Dr. Zhi Zou Copyright: © BioVI Dr. Zhi Zou

Zhi Zou, Diana M. Mate, Maximilian Nöth, Felix Jakob, and Ulrich Schwaneberg*, Chem. - Eur. J., 2020, doi: 10.1002/chem.202002740

Loop engineering and backbone cyclization of sortase A increase the thermal stability and resistance against denaturants.

  Loop engineering of the β6/ β7 loop and backbone cyclizsation of Sa-SrtA increases the robustness of Sa-SrtA against elevated temperatures and denaturing agents (e.g. urea and guanidine hydrochloride). Copyright: © Wiley VCH Loop engineering of the β6/ β7 loop and backbone cyclizsation of Sa-SrtA increases the robustness of Sa-SrtA against elevated temperatures and denaturing agents (e.g. urea and guanidine hydrochloride).

Staphylococcus aureus sortase A (SaSrtA) is widely used for site-specific protein modifications due to its high selectivity and versatility, but it lacks the robustness to perform bioconjugation reactions at elevated temperatures or in the presence of denaturing agents (e.g. urea and guanidine hydrochloride). Loop engineering and subsequent head-to-tail backbone cyclization of SaSrtA was performed to improve thermal robustness and stability against denaturating agents. For activity determination of Sa-SrtA, a cytochrome P450 BM3 (CYP102A1) monooxygenase reconstitution assay was established. Thereby, heme and reductase domain of P450 BM3 were separated in a first step, causing inactivation of P450 BM3. Sa-SrtA enabled the religation of the domains and therefore reconstituted the P450 BM3 activity. Loop engineering and backbone cyclization yielded the cyclized variant CyM6 (rM4-R159N/K162P), that has a 7.5°C increased melting temperature and up to 4.6-fold increased resistance towards urea and guanidine hydrochloride when compared to the parent SaSrtA variant rM4. CyM6 gained up to 2.6-fold (vs parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.