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Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

Linares DM, Alvarez-Sieiro P, del Rio B, Ladero V, Redruello B, Martin MC, Fernandez M, Alvarez MA - Microb. Cell Fact. (2015)

Bottom Line: Lactococcus lactis has been safely consumed in fermented foods for millennia.The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP.The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

View Article: PubMed Central - PubMed

Affiliation: Dairy Research Institute, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Rio Linares s/n, 33300, Villaviciosa, Spain. daniml@ipla.csic.es.

ABSTRACT

Background: Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes.

Results: This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten.

Conclusion: The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

No MeSH data available.


Related in: MedlinePlus

Sensitivity of the ACE system to agmatine. Strength of induction of gfp in L. lactis NZ9000 pACE-GFP. GFP protein activity was determined under a range of agmatine concentrations. Data represent the average of three independent experiments. Bars indicate standard deviations (*p < 0.05)
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Fig3: Sensitivity of the ACE system to agmatine. Strength of induction of gfp in L. lactis NZ9000 pACE-GFP. GFP protein activity was determined under a range of agmatine concentrations. Data represent the average of three independent experiments. Bars indicate standard deviations (*p < 0.05)

Mentions: To verify the usefulness of the ACE system in the expression of heterologous genes, the reporter gene gfp was cloned into the pACE vector under the control of PaguB, thus generating the plasmid pACE-gfp. L. lactis NZ9000—a strain without the AGDI cluster—was transformed with pACE-gfp, thus resulting in L. lactis pACE-gfp. The presence of 10 mM agmatine in a culture of L. lactis pACE-gfp induced the expression of gfp, which was measured in terms of the fluorescence produced (7.87 arbitrary units) (Fig. 3). It should be noted that the expression of gfp was undetectable (<0.5 arbitrary units) in agmatine-uninduced cultures of L. lactis pACE-gfp. Fluorescence was also undetectable (<0.5 arbitrary units) in parallel agmatine-induced cultures of L. lactis NZ9000 harbouring the pACE vector (L. lactis pACE).Fig. 3


Implementation of the agmatine-controlled expression system for inducible gene expression in Lactococcus lactis.

Linares DM, Alvarez-Sieiro P, del Rio B, Ladero V, Redruello B, Martin MC, Fernandez M, Alvarez MA - Microb. Cell Fact. (2015)

Sensitivity of the ACE system to agmatine. Strength of induction of gfp in L. lactis NZ9000 pACE-GFP. GFP protein activity was determined under a range of agmatine concentrations. Data represent the average of three independent experiments. Bars indicate standard deviations (*p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4696319&req=5

Fig3: Sensitivity of the ACE system to agmatine. Strength of induction of gfp in L. lactis NZ9000 pACE-GFP. GFP protein activity was determined under a range of agmatine concentrations. Data represent the average of three independent experiments. Bars indicate standard deviations (*p < 0.05)
Mentions: To verify the usefulness of the ACE system in the expression of heterologous genes, the reporter gene gfp was cloned into the pACE vector under the control of PaguB, thus generating the plasmid pACE-gfp. L. lactis NZ9000—a strain without the AGDI cluster—was transformed with pACE-gfp, thus resulting in L. lactis pACE-gfp. The presence of 10 mM agmatine in a culture of L. lactis pACE-gfp induced the expression of gfp, which was measured in terms of the fluorescence produced (7.87 arbitrary units) (Fig. 3). It should be noted that the expression of gfp was undetectable (<0.5 arbitrary units) in agmatine-uninduced cultures of L. lactis pACE-gfp. Fluorescence was also undetectable (<0.5 arbitrary units) in parallel agmatine-induced cultures of L. lactis NZ9000 harbouring the pACE vector (L. lactis pACE).Fig. 3

Bottom Line: Lactococcus lactis has been safely consumed in fermented foods for millennia.The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP.The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

View Article: PubMed Central - PubMed

Affiliation: Dairy Research Institute, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Rio Linares s/n, 33300, Villaviciosa, Spain. daniml@ipla.csic.es.

ABSTRACT

Background: Lactococcus lactis has been safely consumed in fermented foods for millennia. This Gram-positive bacterium has now become of industrial importance as an expression host for the overproduction of lipopolysaccharide-free recombinant proteins used as food ingredients, therapeutic proteins and biotechnological enzymes.

Results: This paper reports an agmatine-controlled expression (ACE) system for L. lactis, comprising the lactococcal agmatine-sensor/transcriptional activator AguR and its target promoter P(aguB). The usefulness and efficiency of this system was checked via the reporter gene gfp and by producing PEP (Myxococcus xanthus prolyl-endopeptidase), an enzyme of biomedical interest able to degrade the immunotoxic peptides produced during the gastrointestinal breakdown of gluten.

Conclusion: The ACE system developed in this work was suitable for the efficient expression of the functional recombinant proteins GFP and PEP. The expression system was tightly regulated by the agmatine concentration and allowed high protein production without leakiness.

No MeSH data available.


Related in: MedlinePlus