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.
Highlights
Functionalization of proteins is often performed by targeting reactive endogenous amino acid side chains (e.g. -NH2 in lysine or -SH in cysteine residues).[1]
The resulting protein-sortase thioester is attacked by the amino group of an N-terminal glycine residue of protein 2 (P2), resulting in a protein conjugate of protein 1 and protein 2 (Scheme 1).[14]
Given to the comparably high transpeptidase activity among the sortases,[21] Staphylococcus aureus sortase A (SaSrtA) is most frequently used in sortagging applications.[22]
Summary
Functionalization of proteins is often performed by targeting reactive endogenous amino acid side chains (e.g. -NH2 in lysine or -SH in cysteine residues).[1]. A few studies reported the engineering of SaSrtA WT for increased stability towards thermal and chemical denaturation (e.g. 11.2 8C improvement in melting temperature (Tm) and 4.5-fold resistance in presence of 2.5 m urea).[31,32] engineering of highly active SaSrtA variants with enhanced tolerance against thermal and chemical stress has not been reported yet.
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