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Expression and purification of the native C‐amidated antimicrobial peptide maculatin 1.1
Author(s) -
Zhu Shiying,
Weber Daniel K.,
Separovic Frances,
Sani MarcAntoine
Publication year - 2021
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.3330
Subject(s) - peptide , intein , escherichia coli , antimicrobial peptides , antimicrobial , recombinant dna , biochemistry , bacteria , chemistry , peptide sequence , magainin , nuclear magnetic resonance spectroscopy , in vivo , circular dichroism , combinatorial chemistry , biology , stereochemistry , microbiology and biotechnology , rna , gene , genetics , rna splicing
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.