title: Expression, purification, and micelle reconstitution of antimicrobial piscidin 1 and piscidin 3 for NMR studies.
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 Escherichiacoli 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.
authors.affiliation: Department of Biological Chemistry & Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemistry, Hamilton College, 198 College Hill Road, Clinton, NY 13323, United States. Electronic address: mcotten@hamilton.edu.
authors.forename: Myriam L
authors.initials: ML
authors.lastname: Cotten
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D023181:Antimicrobial Cationic Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D001492:Bass / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
chemical_list: D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; D008823:Micelles; C449802:moronecidin protein, Morone saxatilis; D012967:Sodium Dodecyl Sulfate
keywords: (13)C/(15)N uniformly labeled; Amphipathic antimicrobial peptides; Membrane-active peptides; Micelles; NMR; Piscidin