title: 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.
abstract: 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.
authors.affiliation: 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.
authors.forename: Sara
authors.identifier: 0000-0001-9889-3692
authors.initials: S
authors.lastname: Arbulu
authors.affiliation: 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.
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Jiménez
authors.affiliation: 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.
authors.forename: Loreto
authors.initials: L
authors.lastname: Gútiez
authors.affiliation: 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.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Cintas
authors.affiliation: 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.
authors.forename: Carmen
authors.initials: C
authors.lastname: Herranz
authors.affiliation: 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.
authors.forename: Pablo E
authors.initials: PE
authors.lastname: Hernández
mesh_terms: D001430:Bacteriocins / Q000096:biosynthesis* / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D005813:Genes, Synthetic / Q000235:genetics*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D001430:Bacteriocins; D011994:Recombinant Proteins
references.citation: PLoS One. 2012;7(9):e45848
references.pmid: 23029273
references.citation: Mol Biotechnol. 2000 Sep;16(1):23-52
references.pmid: 11098467
references.citation: Chembiochem. 2014 Jan 24;15(2):243-9
references.pmid: 24382692
references.citation: Int J Microbiol. 2012;2012:386410
references.pmid: 22187559
references.citation: Yeast. 2008 Jan;25(1):1-8
references.pmid: 17973232
references.citation: J Food Prot. 2005 Jul;68(7):1450-3
references.pmid: 16013385
references.citation: Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2384-9
references.pmid: 17284603
references.citation: Appl Environ Microbiol. 2012 Aug;78(16):5956-61
references.pmid: 22685156
references.citation: PLoS One. 2012;7(6):e37915
references.pmid: 22685548
references.citation: Poult Sci. 2010 Aug;89(8):1763-8
references.pmid: 20634535
references.citation: Mol Biotechnol. 2014 Jun;56(6):571-83
references.pmid: 24510220
references.citation: Appl Microbiol Biotechnol. 2000 May;53(5):575-82
references.pmid: 10855718
references.citation: Appl Environ Microbiol. 2011 Jan;77(1):369-73
references.pmid: 21057028
references.citation: Anal Chem. 2000 May 1;72(9):2132-41
references.pmid: 10815976
references.citation: J Ind Microbiol Biotechnol. 2013 Sep;40(9):977-93
references.pmid: 23794087
references.citation: Appl Environ Microbiol. 2008 Apr;74(8):2471-9
references.pmid: 18310424
references.citation: Nat Rev Microbiol. 2013 Feb;11(2):95-105
references.pmid: 23268227
references.citation: Science. 1999 Dec 17;286(5448):2361-4
references.pmid: 10600751
references.citation: Proteomics. 2009 Oct;9(20):4632-41
references.pmid: 19743430
references.citation: Antimicrob Agents Chemother. 2006 Sep;50(9):3111-6
references.pmid: 16940109
references.citation: Antimicrob Agents Chemother. 2008 Mar;52(3):1094-100
references.pmid: 18086839
references.citation: Mol Membr Biol. 2011 Sep;28(6):398-411
references.pmid: 21770695
references.citation: J Food Prot. 2005 Jan;68(1):11-7
references.pmid: 15690798
references.citation: Microbiol Mol Biol Rev. 2006 Jun;70(2):564-82
references.pmid: 16760314
references.citation: Appl Environ Microbiol. 2011 Apr;77(8):2749-54
references.pmid: 21378051
references.citation: Appl Environ Microbiol. 2005 May;71(5):2310-7
references.pmid: 15870316
references.citation: J Biotechnol. 2011 Oct 20;156(1):76-86
references.pmid: 21839785
references.citation: J Agric Food Chem. 2008 Mar 26;56(6):1942-8
references.pmid: 18293921
references.citation: J Biomed Biotechnol. 2005;2005(4):374-84
references.pmid: 16489270
references.citation: PLoS One. 2013;8(9):e75347
references.pmid: 24069404
references.citation: Appl Microbiol Biotechnol. 2007 Dec;77(3):513-23
references.pmid: 17924105
references.citation: Methods Mol Biol. 2012;866:11-23
references.pmid: 22454110
references.citation: J Clin Microbiol. 2008 Nov;46(11):3863-5
references.pmid: 18768652
references.citation: Peptides. 2012 Dec;38(2):446-56
references.pmid: 23022589
references.citation: Microb Cell Fact. 2008 Apr 04;7:11
references.pmid: 18394160
references.citation: Biochim Biophys Acta. 2010 Jun;1803(6):694-705
references.pmid: 20219571
references.citation: Appl Microbiol Biotechnol. 2014 Jun;98(12):5301-17
references.pmid: 24743983
references.citation: Microbiology. 2011 Dec;157(Pt 12):3256-67
references.pmid: 21980118
references.citation: Antimicrob Agents Chemother. 2005 Jul;49(7):3004-8
references.pmid: 15980385
references.citation: Nat Rev Microbiol. 2005 Oct;3(10):777-88
references.pmid: 16205711
references.citation: J Am Chem Soc. 2012 Dec 5;134(48):19540-3
references.pmid: 23167271