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. 2021:2355:83-92.
doi: 10.1007/978-1-0716-1617-8_8.

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

Poanna Tran  1 , Christina I Schroeder  2   3
Affiliations

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

Poanna Tran et al. Methods Mol Biol. 2021.

Abstract

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.

Keywords: Disulfide-rich peptide; Double-knotted peptide; Enzymatic ligation; Site-specific ligation; Sortase A; Transpeptidase.

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Figures

Fig. 1
Fig. 1
Mechanism of sortase A ligation of two disulfide-rich peptides
Fig. 2
Fig. 2
General procedure for the production of a bivalent peptide with sortase A
Fig. 3
Fig. 3
LC/MS traces showing the SrtA5°-mediated ligation of [G5]KIIIA and PaurTx3[LPATGG] after 15 min. (Adapted from Tran et al. [12])
See this image and copyright information in PMC

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