title: Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli.
abstract: The cloning and expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, was studied in Escherichia coli. PCR-amplified products of the preenterocin P gene (entP) or entP plus the putative EntP immunity gene (entiP), were cloned in plasmid pETBlue-1 under the control of the inducible T7lac promoter. Although target genes in derivative plasmids pJG01 (entP) and pJG02 (entP plus entiP) did not generate products with antimicrobial activity after an in vitro combined transcription/translation reaction, they were expressed as biologically active products following transformation and induction in the E. coli Tuner(DE3)pLacI host. The use of specific antibodies and an ELISA permitted the detection and quantification of EntP in the supernatant (SN), cellular soluble protein fraction (CSF), and inclusion bodies (IB) of E. coli Tuner(DE3)pLacI cells transformed with either pJG01 or pJG02. Functional EntP from the supernatants of E. coli Tuner(DE3)pLacI (pJG01) cultures grown in a complex medium was recovered, at a high efficiency, by immunoaffinity chromatography in a single step. A purification method based on hydrophobic adsorption and reverse-phase chromatographies also permitted the recovery of active EntP from the supernatants of the same cultures grown in a minimally defined medium. The E. coli Tuner(DE3)pLacI (pJG01) cells would merit consideration as an alternative experimental model for the heterologous production and functional expression of EntP, as well as for the fast and efficient recovery of this bacteriocin from the supernatant of this recombinant producer.
authors.affiliation: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Gutiérrez
authors.forename: R
authors.initials: R
authors.lastname: Criado
authors.forename: R
authors.initials: R
authors.lastname: Citti
authors.forename: M
authors.initials: M
authors.lastname: Martín
authors.forename: C
authors.initials: C
authors.lastname: Herranz
authors.forename: I F
authors.initials: IF
authors.lastname: Nes
authors.forename: L M
authors.initials: LM
authors.lastname: Cintas
authors.forename: P E
authors.initials: PE
authors.lastname: Hernández
mesh_terms: D001430:Bacteriocins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D010957:Plasmids
chemical_list: D001430:Bacteriocins; D004269:DNA, Bacterial; C417717:enterocin P