Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris.
We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid seque...
| Publicado en: | BioMed Research International Vol. 2015; pp. 1 - 12 |
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| Autores principales: | , , , , , |
| Formato: | research tables/charts Journal Article |
| Publicado: |
Wiley-Blackwell
3/2/2015
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=109273545&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 109273545 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 23146133 FT2T jtl: BioMed Research International issn: 23146133 maglogo: N pubinfo: dt: 3/2/2015 vid: 2015 pid: 480 pub: Wiley-Blackwell place: Malden, Massachusetts artinfo: ui: 109273545 109273545 109273545 10.1155/2015/767183 109273545 ppf: 1 ppct: 11 formats: fmt: @attributes: type: P tig: atl: Cloning and Expression of Synthetic Genes Encoding the Broad Antimicrobial Spectrum Bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by Recombinant Pichia pastoris. aug: au: Arbulu, Sara Jiménez, Juan J. Gútiez, Loreto Cintas, Luis M. Herranz, Carmen Hernández, Pablo E. affil: Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid (UCM), Avenida Puerta de Hierro, s/n, 28040 Madrid, Spain sug: subj: Bacterial Proteins Genetic Techniques Recombinant Proteins Therapeutic Use Funding Source Academic Medical Centers Spain Polymerase Chain Reaction ab: We have evaluated the cloning and functional expression of previously described broad antimicrobial spectrum bacteriocins SRCAM 602, OR-7, E-760, and L-1077, by recombinant Pichia pastoris. Synthetic genes, matching the codon usage of P. pastoris, were designed from the known mature amino acid sequence of these bacteriocins and cloned into the protein expression vector pPICZαA. The recombinant derived plasmids were linearized and transformed into competent P. pastoris X-33, and the presence of integrated plasmids into the transformed cells was confirmed by PCR and sequencing of the inserts. The antimicrobial activity, expected in supernatants of the recombinant P. pastoris producers, was purified using a multistep chromatographic procedure including ammonium sulfate precipitation, desalting by gel filtration, cation exchange-, hydrophobic interaction-, and reverse phase-chromatography (RP-FPLC). However, a measurable antimicrobial activity was only detected after the hydrophobic interaction and RP-FPLC steps of the purified supernatants. MALDI-TOF MS analysis of the antimicrobial fractions eluted from RP-FPLC revealed the existence of peptide fragments of lower and higher molecular mass than expected. MALDI-TOF/TOF MS analysis of selected peptides from eluted RP-FPLC samples with antimicrobial activity indicated the presence of peptide fragments not related to the amino acid sequence of the cloned bacteriocins. pubtype: Academic Journal doctype: research tables/charts Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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