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OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

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Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Zhi Zou^1,2, Diana M Mate^2,3, Maximilian Nöth^1,2

¹Institute of Biotechnology, RWTH Aachen University, Worringerweg 3, 52074, Aachen, Germany.

Chemistry (Weinheim an Der Bergstrasse, Germany)

|July 11, 2020

Summary

This summary is machine-generated.

A novel cyclized Staphylococcus aureus sortase A (SaSrtA) variant, CyM6, enhances protein bioconjugation. This engineered enzyme exhibits improved stability and higher yields under challenging denaturing conditions.

Keywords:

biocatalysis protein engineering site-specificity sortase A thermal stability

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Area of Science:

Biochemistry
Protein Engineering
Enzymology

Background:

Staphylococcus aureus sortase A (SaSrtA) is a key enzyme for site-specific protein modification.
Existing SaSrtA variants have limited stability at elevated temperatures and in denaturing agents, restricting their application scope.

Purpose of the Study:

To engineer a more robust SaSrtA variant for improved bioconjugation efficiency.
To enhance the thermal and chemical stability of SaSrtA for broader applications.

Main Methods:

Loop engineering of SaSrtA to introduce structural modifications.
Head-to-tail backbone cyclization to create a stabilized enzyme variant (CyM6).
Comparative analysis of CyM6 and parent rM4 stability and catalytic activity.

Main Results:

The cyclized variant CyM6 demonstrated a 7.5°C increase in melting temperature compared to the parent rM4.
CyM6 exhibited up to 4.6-fold enhanced resistance to denaturing agents.
Under denaturing conditions, CyM6 achieved up to 2.6-fold higher yields in peptide and primary amine ligation.

Conclusions:

Engineered SaSrtA variant CyM6 possesses significantly improved thermal and chemical stability.
CyM6 enhances the efficiency of site-specific protein bioconjugation, particularly under harsh conditions.
This stabilized sortase A variant expands the utility of sortase-mediated ligation in biotechnology and protein engineering.