Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1.

Shiying Zhu, Daniel K Weber, Frances Separovic, Marc-Antoine Sani

Journal of peptide science : an official publication of the European Peptide Society 2021 Aug

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Maculatin 1.1 (Mac1) is an antimicrobial peptide (AMP) from an Australian tree frog and exhibits low micromolar activity against Gram-positive bacteria. The antimicrobial properties of Mac1 are linked to its disruption of bacterial lipid membranes, which has been studied extensively by in vitro nuclear magnetic resonance (NMR) spectroscopy and biophysical approaches. Although in vivo NMR has recently proven effective in probing peptide-lipid interplay in live bacterial cells, direct structural characterisation of AMPs has been prohibited by low sensitivity and overwhelming background noise. To overcome this issue, we report a recombinant expression protocol to produce isotopically enriched Mac1. We utilized a double-fusion construct to alleviate toxicity against the Escherichia coli host and generate the native N-free and C-amidated termini Mac1 peptide. The SUMO and intein tags allowed native N-terminus and C-terminal amidation, respectively, to be achieved in a one-pot reaction. The protocol yielded 0.1 mg/L of native, uniformly 15 N-labelled, Mac1, which possessed identical structure and activity to peptide obtained by solid-phase peptide synthesis. © 2021 European Peptide Society and John Wiley & Sons, Ltd.

Citation

Shiying Zhu, Daniel K Weber, Frances Separovic, Marc-Antoine Sani. Expression and purification of the native C-amidated antimicrobial peptide maculatin 1.1. Journal of peptide science : an official publication of the European Peptide Society. 2021 Aug;27(8):e3330