Production of active pediocin PA-1 in Escherichia coli using a thioredoxin gene fusion expression approach: cloning, expression, purification, and characterization

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Abstract:

Antimicrobial peptides possess cationic and amphipathic properties that allow for interactions with the membrane of living cells. Bacteriocins from lactic acid bacteria, in particular, are currently being studied for their potential use as food preservatives and for applications in health care. However, bacteriocin exploitation is often limited owing to low production yields. Gene cloning and heterologous protein or peptide production is one way to possibly achieve overexpression of bacteriocins to support biochemical studies. In this work, production of recombinant active pediocin PA-1 (PedA) was accomplished in Escherichia coli using a thioredoxin (trx) gene fusion (trxpedA) expression approach. Trx–PedA itself did not show any biological activity, but upon cleavage by an enterokinase, biologically active pediocin PA-1 was obtained. Recombinant pediocin PA-1 characteristics (molecular mass, biological activity, physicochemical properties) were very similar to those of native pediocin PA-1. In addition, a 4- to 5-fold increase in production yield was obtained, by comparison with the PA-1 produced naturally by Pediococcus acidilactici PAC 1.0. The new production method, although not optimized, offers great potential for supporting further investigations on pediocin PA-1 and as a first-generation process for the production of pediocin PA-1 for high-value applications.

Les peptides antimicrobiens possèdent des propriétés cationiques et amphipathiques leur permettant d’interagir avec la membrane des organismes vivants. Les bactériocines dérivées des bactéries lactiques, en particulier, sont présentement étudiées dû à leur potentiel comme agent de conservation alimentaire et pour des applications possibles en santé. Toutefois, leur utilisation est souvent limitée par un faible niveau de production. Les techniques de clonage et de production de peptides ou protéines hétérologues offrent une voie permettant d’obtenir possiblement la sur-production de bactériocines, laquelle est requise afin de supporter diverses études biochimiques. Dans cette étude, nous décrivons la production de pédiocine PA-1 recombinante et biologiquement active par Escherichia coli, grâce à une stratégie de fusion du gène pedA avec le gène de la thiorédoxine. Le premier produit de l’expression du gène de fusion (trxpedA), la protéine de fusion Trx-PedA, ne montrait aucune activité biologique mais, après clivage à l’entérokinase, la pédiocine PA-1 mature et biologiquement active fut obtenue. La pédiocine PA-1 recombinante montre des propriétés (masse moléculaire, activité biologique, caractéristiques physico-chimiques) très semblables à celle de la pédiocine PA-1 naturelle. En plus, une augmentation de 4 à 5 fois de la production de pédiocine, bien que non optimisée, offre beaucoup de potentiel non seulement afin de supporter des travaux additionnels sur la pédiocine PA-1 mais aussi comme procédé de production de première génération pour la production de pédiocine PA-1 pour des applications à forte valeur ajoutée.

Document Type: Research Article

Publication date: November 1, 2007

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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