Enzymatic Ligation of Disulfide-Rich Animal Venom Peptides: Using Sortase A to Form Double-Knotted Peptides.

Sortase A is a thiol transpeptidase expressed by Gram-positive bacteria. This enzyme is capable of site-specifically ligating peptides containing the C-terminal recognition motif LPXTG to peptides containing an N-terminal polyglycine sequence, forming a native peptide bond. Here, we describe the preparation and application of sortase A to the ligation of two individually folded disulfide-rich animal venom peptides in order to form a heterodimeric double-knotted peptide with a native peptide linker. This method is mild enough to preserve the structures and disulfide connectivities of the peptides during ligation. We employed a highly efficient sortase A pentamutant (SrtA5°), which brings the reaction to completion within 15 min with a ~50-80% yield of ligated peptide.