Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Expression, purification, and micelle reconstitution of antimicrobial Piscidin 1 and Piscidin 3 for NMR studies.
Protein Expr Purif. 2014 Aug 12;
Authors: Chen W, Cotten ML
Abstract
Piscidin 1 and piscidin 3, which were discovered in the mast cells of hybrid striped sea bass, are homologous antimicrobial peptides that are active against drug-resistant bacteria. Piscidin 1, the more antimicrobial and hemolytic peptide, also has anti-HIV-1 and anti-cancer properties. To understand the reasons underlying the different biological activities of the two peptides and identify principles to design antimicrobial drugs with improved efficacy and lower toxicity, their atomic-level structures must be obtained under physiologically-relevant conditions. High-resolution backbone structures of both piscidins exist in the presence of hydrated phospholipid bilayers but full structures that include the side chains are missing. Here, the piscidins 1 and 3 genes were cloned into the TrpLE vector. The corresponding TrpLE-piscidin fusion partners were expressed in E. Coli and recovered from inclusion bodies. Following steps that included Ni-NTA chromatography, cyanogen bromide cleavage of the fusion proteins, and reverse-phase HPLC, purified piscidins 1 and 3 were recovered in very good yield and characterized by NMR. High quality (15)N-(1)H HSQC spectra of piscidins 1 and 3 bound to SDS micelles were collected, demonstrating the feasibility of producing and purifying the isotopically-labeled piscidin peptides required to determine their full structures by multidimensional NMR spectroscopy.
PMID: 25131859 [PubMed - as supplied by publisher]
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