Sortase-Mediated Protein Ligation:  A New Method for Protein Engineering

Abstract

Sortase (SrtA), a transpeptidase from Staphylococcus aureus, catalyzes a cell-wall sorting reaction at an LPXTG motif by cleaving between threonine and glycine and subsequently joining the carboxyl group of threonine to an amino group of pentaglycine on the cell wall peptidoglycan. We have applied this transpeptidyl activity of sortase to in vitro protein ligation. We found that in the presence of sortase, protein/peptide with an LPXTG motif can be specifically ligated to an aminoglycine protein/peptide via an amide bond. Additionally, sortase can even conjugate substrates such as (d)-peptides, synthetic branched peptides, and aminoglycine-derivatized small molecules to the C terminus of a recombinant protein. The sortase-mediate protein ligation is robust, specific, and easy to perform, and can be widely applied to specific protein conjugation with polypeptides or molecules of unique biochemical and biophysical properties.

Butelase 1: A Versatile and Multi-Purpose Ligase

“…The second approach is the C-terminal-specific ligation. It requires the use of an enzyme, a ligase, to recognize a specific amino acid at or near the C-terminus [7]. This approach is highly underdeveloped because naturally occurring ligases are rare and our discovery of butelase 1 as an Asx-specific ligase provides a promising tool to fill this void.…”

Backbone cyclization of a recombinant cystine‐knot peptide by engineered Sortase A

“…We selected the enzyme Sortase A, a transpeptidase from Staphylococcus aureus, which recognizes a consensus peptide sequence Leu-Pro-X-Thr-Gly (X = any amino acid residue). This enzyme cleaves the amide bond between Thr and Gly and catalyzes the ligation of the cleaved sequence to a Gly-Gly-Gly motif, resulting in a contiguous LeuPro-X-Thr-Gly-Gly-Gly polypeptide chain [20][21][22]. The Sortase A reaction which occurs primarily as an intermolecular reaction in S. aureus, has been recently exploited for generating circular proteins or creating fusions of different proteins, as well as labeling proteins at the N-or C-termini [23][24][25].…”

Evolution of amide bond formation

“…A sortase-mediated ligation of proteins to both peptides and non-peptides was demonstrated in vitro to be a new method for protein engineering. 98 As mentioned earlier (3.1), azides are involved in the synthesis of proteins by native chemical ligation (NCL). A modification produced the amide bond between the two coupling partners without an intervening triaryl phosphine oxide and is referred to as the "traceless" Staudinger reaction.…”