title: Expression of an optimized Argopecten purpuratus antimicrobial peptide in E. coli and evaluation of the purified recombinant protein by in vitro challenges against important plant fungi.
abstract: Antimicrobial peptides (AMP) have been widely described in several organisms from different kingdoms. We recently designed and evaluated a synthetic version of an AMP isolated and characterized from Argopecten purpuratus hemocytes. This study describes the generation of a chimaeric gene encoding for Ap-S, the use of this construct to transform E. coli strain BL21, and the evaluation of the purified recombinant Ap-S (rApS) as an antifungal agent. The proposed gene coding for rAp-S consists of 93 nucleotides arranged downstream from the IPTG-inducible T7 promoter. The best synthesis conditions were obtained after E. coli cultivation at 26°C for 3h, which allowed for the production of an rAp-S-enriched fraction containing the peptide at 249μM. Mass spectrometry analysis of the purified rAp-S (3085.80Da) showed the addition of a glycine residue on its N-terminal end derived from vector design and peptide purification. The purified rApS fraction was assayed for antifungal activity by direct addition of purified rApS elution to potato dextrose agar media at a final concentration of 81nM. These assays showed important growth inhibitions of both biotrophic (Fusarium oxysporum, Trichoderma harzianum) and necrotrophic (Botrytis cinerea, Alternaria spp.) fungi in that the hyphae structures and spore count were affected in all cases. The strategy of cloning and expressing rAp-S in E. coli, the high yield obtained and its successful use for controlling pathogenic fungi suggest that this molecule could be applied to agricultural crops using various management strategies.
authors.affiliation: Biotechnology Doctoral Program, Universidad Técnica Federico Santa María-Pontificia Universidad Católica de Valparaíso, Av. Brasil 2950, Valparaíso, Chile.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Tapia
authors.forename: Christian
authors.initials: C
authors.lastname: Montes
authors.forename: Patricia
authors.initials: P
authors.lastname: Rebufel
authors.forename: Alberto
authors.initials: A
authors.lastname: Paradela
authors.forename: Humberto
authors.initials: H
authors.lastname: Prieto
authors.forename: Gloria
authors.initials: G
authors.lastname: Arenas
mesh_terms: D000528:Alternaria / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000302:isolation & purification / Q000494:pharmacology*; D001483:Base Sequence; D020171:Botrytis / Q000187:drug effects; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005670:Fusarium / Q000187:drug effects; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D049897:Pectinidae / Q000737:chemistry*; D010455:Peptides / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014242:Trichoderma / Q000187:drug effects
chemical_list: D000935:Antifungal Agents; D010455:Peptides; D011994:Recombinant Proteins