Efficient N‐Terminal Labeling of Proteins by Use of Sortase

From protein total synthesis to peptide transamidation and metathesis: playing with the reversibility of N,S-acyl or N,Se-acyl migration reactions

“…A gentler alternative approach is to activate the amide bond using biocatalysts such as proteases or peptidases in waterbased solvent systems [7,8]. However, avoiding the native hydrolytic reaction catalyzed by these enzymes is an issue.…”

“…However, avoiding the native hydrolytic reaction catalyzed by these enzymes is an issue. Moreover, this mode of activation is either too specific for a given peptide recognition motif, for example Leu-Pro-X-Thr-Gly for sortase A (X is any amino acid) [7], or is poorly selective for a given peptide bond. The transamidation or amide metathesis reactions of simple amides can also be catalyzed by metal complexes [6 ].…”

Sortagging: A Robust and Efficient Chemoenzymatic Ligation Strategy

“…One disadvantage of this strategy is the necessity to use a nonstandard dipeptide. [114,115] Replacing the C-terminal glycine residue by a methyl ester is a comparable procedure established by Ploegh et al However, stoichiometric quantities of the enzyme and excess methyl ester are necessary to obtain reasonable yields. [43] Mezo et al prepared LPXTG substrates that produce SrtA-excised peptide fragments that deactivate through diketopiperazine formation (Figure 9 C).…”

“…The latter is no substrate for the reverse reaction as proven by different conversions of the depsipeptide and the corresponding normal peptide. [114,115] C) LPETGGX substrates that contain a C-terminal N-acetylated serine (n = 1) or homoserine (n = 2) produce SrtA-excised peptide fragments that readily convert to 2,5-diketopiperazine and the corresponding N-acetylated amino acid. [116] D) Sortase-mediated hydrazinolysis is an irreversible chemoenzymatic ligation method that utilizes N-terminal hydrazides.…”

Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins

“…Recent methodologies to address this issue of irreversibility have included the use of a sortase-tagged expressed protein ligation (STEPL) system that circumvents the removal of unconjugated species [12], dialysis to remove reaction by-products [13] and the introduction of tryptophan-derived zippers around the SrtA recognition motif that induces the formation of a stable β-hairpin [14]. Other research groups have solved this problem by utilizing a depsipeptide, which replaces the amide bond between the threonine and glycine residues with an ester linkage [15]. These challenges make protein modification using sortase cumbersome and potentially expensive when the fluorophore-containing peptide is needed in many fold excess.…”