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

Author:

Tran, Poanna

Schroeder,Christina
Author Address

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia., Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia. christina.schroeder@nih.gov., National Cancer Institute, National Institutes of Health, Frederick, MD, USA. christina.schroeder@nih.gov.,

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. © 2021. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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