Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilisation

Abstract

Sortases are transpeptidases produced by Gram-positive bacteria to anchor cell surface proteins covalently to the cell wall. The Staphylococcus aureus sortase A (SrtA) cleaves a short C-terminal recognition motif (LPXTG) on the target protein followed by the formation of an amide bond with the pentaglycine cross-bridge in the cell wall. Over recent years, several researchers have exploited this specific reaction for a range of biotechnology applications, including the incorporation of non-native peptides and non-peptidic molecules into proteins, the generation of nucleic acid-peptide conjugates and neoglycoconjugates, protein circularisation, and labelling of cell surface proteins on living cells.

Reprogramming the specificity of sortase enzymes

“…The bacterial transpeptidase Staphylococcus aureus sortase A (SrtA) mediates the anchoring of proteins to the bacterial cell wall and has been widely used in bioconjugate synthesis (5). Wild-type SrtA binds a small, five-amino acid "sorting motif" (Leu-Pro-XThr-Gly, LPXTG, where X = any amino acid) and cleaves the scissile Thr-Gly peptide bond via a cysteine protease-like mechanism, resulting in loss of the C-terminal glycine to yield a thioacyl intermediate.…”

Protein trans-splicing as a protein ligation tool to study protein structure and function

“…Although subtiligase has been successfully used for the total synthesis of ribonuclease A (51) and a few other proteins (52), this ligase has never become widely used. Recently, sortase A from Staphylococcus aureus, which covalently attaches proteins to the cell wall, has been used for ligation of two polypeptide chains wfor a review, see (53)x. Though promising alternatives, the slow reaction kinetics and necessity to use excess enzyme represent a bottleneck for the use of subtiligase and sortase as protein ligases in a wide range of applications.…”

Sortase-mediated backbone cyclization of proteins and peptides

“…This leads to the formation of a covalent thioester bond between sortase and its substrate with concomitant breaking of the threonine-glycine peptide bond. This thioester bond is very stable to hydrolysis by water molecules but is readily resolved by nucleophilic attack of a primary amine attached to a primary carbon, for instance the Cα atom of glycine (Proft, 2010). Isopeptide ligation, i.e., coupling to the ε-amino group of lysines, does not generally occur, because at physiological pH this group is protonated, resulting in decreased nucleophilicity.…”

“…(iii) This leads to the formation of a covalent thioester bond between sortase and its substrate with concomitant breaking of the threonine-glycine peptide bond and release of glycine. This thioester bond is very stable to hydrolysis by water molecules but is readily resolved by nucleophilic attack of a suitably exposed primary amine, e.g., the N-terminus of a glycine residue (figure adapted from Proft, 2010).…”

“…As a result, only a small stretch of six amino acids, LPXTGG, ends up as 'contamination' between the two conjugation partners, reducing the risk on disturbance of the protein structure or unwanted biological side effects due to ligation. Together with the easy access to large quantities of bioactive recombinant sortase A, this versatility has made this enzyme a true workhorse in the fields of site-specific modification and functionalization of proteins (sortagging) and glycopeptides, site-specific ligation of peptides and proteins, and covalent immobilization of proteins, which contributed significantly to the scientific progress in these disciplines over the last decade (Proft, 2010;Wu and Guo, 2012).…”