title: Stable expression of bioactive recombinant pleurocidin in a fish cell line.
abstract: Pleurocidin (Ple), a linear cationic peptide of 25 amino acids, is a member of a larger family of antimicrobial peptides present in flatfish. Previous studies have shown that Ple displays a strong antimicrobial activity against a broad spectrum of bacteria and appears to play a role in innate host defence. In this work, the genomic sequence encoding the Ple prepropeptide has been isolated from Limanda limanda and cloned in a vector under the control of a non-viral promoter (the carp beta-actin promoter). By using this construction, expression of bioactive Ple was demonstrated in transformed fish cell lines continuously growing for more than 2 years. Furthermore, the study of Ple processing, maturation and secretion (by using fusion with green fluorescence protein) and the high bactericidal activity of the secreted recombinant Ple (detectable in cell supernatants without any concentration) are all reported here, as no other recombinant Ple or fish antimicrobial peptide have been expressed before to that extent. Such an overexpression of recombinant Ple or any other related antimicrobial peptide might improve the chances to develop new antibiotic agents, as well as to provide essential information about the mechanism of action, range of activity and the role in the innate immune response of antibiotic peptides.
authors.affiliation: Instituto de Biologia Molecular y Celular (IBMC), Miguel Hernández University, 03202 Elche, Spain.
authors.forename: I
authors.initials: I
authors.lastname: Brocal
authors.forename: A
authors.initials: A
authors.lastname: Falco
authors.forename: V
authors.initials: V
authors.lastname: Mas
authors.forename: A
authors.initials: A
authors.lastname: Rocha
authors.forename: L
authors.initials: L
authors.lastname: Perez
authors.forename: J M
authors.initials: JM
authors.lastname: Coll
authors.forename: A
authors.initials: A
authors.lastname: Estepa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000187:drug effects; D029941:Fish Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D005412:Flatfishes / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism; D017686:Oncorhynchus mykiss; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis
chemical_list: D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; C105851:pleurocidin; D049452:Green Fluorescent Proteins