Stable expression of bioactive recombinant pleurocidin in a fish cell line

• Published in: Applied microbiology and biotechnology Vol. 72; no. 6; pp. 1217 - 1228
• Main Authors: Brocal, I, Falco, A, Mas, V, Rocha, A, Perez, L, Coll, J. M, Estepa, A
• Format: Journal Article
• Language: English
• Published: Berlin Berlin/Heidelberg : Springer-Verlag 01-10-2006; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amino acids; Animals; Antibiotics; Antimicrobial agents; Antimicrobial Cationic Peptides - biosynthesis; Antimicrobial Cationic Peptides - genetics; Antimicrobial Cationic Peptides - pharmacology; Bacteria; Base Sequence; Biological and medical sciences; Biotechnology; Cell Line, Transformed; Cell Survival - drug effects; Cloning, Molecular; Escherichia coli - drug effects; Fish Proteins - biosynthesis; Fish Proteins - genetics; Fish Proteins - pharmacology; Flatfishes - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Immune response; Immune system; Limanda limanda; Marine; Molecular Sequence Data; Muscle, Skeletal - metabolism; Oncorhynchus mykiss; Peptides; Recombinant Fusion Proteins - biosynthesis; Cell line
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-006-0393-7

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 β-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.