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Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli.

Nasr R, Akbari Eidgahi MR - Jundishapur J Microbiol (2014)

Bottom Line: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes.In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins.Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.

View Article: PubMed Central - PubMed

Affiliation: Semnan Biotechnology Research Center, Semnan University of Medical Sciences, Semnan, IR Iran.

ABSTRACT

Background: Anaerobic-inducible promoters are alternatives of chemical-inducible promoters for expression of recombinant proteins especially in conditions where chemical induction is not possible or anaerobic conditions are preferable. The nirB promoter is the promoter of the first gene of nir operon in Escherichia coli, which encodes NADH-dependent nitrite reductase. This promoter is naturally induced under anaerobic conditions and upregulated by nitrite and nitrate.

Objectives: The current study was carried out to construct a synthetic nirB promoter that does not respond to chemical inducers (nitrite or nitrate), but instead responds to anaerobic induction. For this purpose, a new plasmid was constructed (pFSnirB78-23LTB), which contains a synthetic nirB promoter. The activity of this plasmid was evaluated in E. coli under both aerobic and anaerobic conditions and in response to chemical inducers, nitrite and nitrate.

Materials and methods: A synthetic nirB promoter was firstly cloned into a pKK223 derivative plasmid and then the heat labile toxin B subunit gene (LTB) of entrotoxigenic E. coli was cloned under the control of this promoter. The inducibility of this plasmid in E. coli was measured under anaerobic conditions in the presence or absence of nitrite or nitrate by ganglioside GM1 ELISA.

Results: Our data showed that this promoter is strongly induced under anaerobic conditions while it showed much lower activity (11%) under aerobic conditions. In contrast to the native promoter, this promoter was not induced by chemical inducers, nitrite or nitrate.

Conclusions: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes. In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins. Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.

No MeSH data available.


Related in: MedlinePlus

Schematic Construction of pFSnirB78-23LTB Plasmid
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fig12084: Schematic Construction of pFSnirB78-23LTB Plasmid

Mentions: In this study, we constructed a new plasmid by two-step cloning as shown in Figure 1. At first, we cloned the 78 bp synthetic nirB promoter in a pKK223 derivative plasmid and then the LTB gene (375 bp) was cloned under the control of this promoter and the final plasmid was named pFSnirB78 -23LTB. The second codon of the native LTB gene was changed from AAT to GAT, which altered Asn to Asp in the second amino acid of the LTB signal peptide. The plasmid was transformed into E. coli DH5 in order to measure promotor activity following restriction analysis of the plasmid.


Construction of a Synthetically Engineered nirB Promoter for Expression of Recombinant Protein in Escherichia coli.

Nasr R, Akbari Eidgahi MR - Jundishapur J Microbiol (2014)

Schematic Construction of pFSnirB78-23LTB Plasmid
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216584&req=5

fig12084: Schematic Construction of pFSnirB78-23LTB Plasmid
Mentions: In this study, we constructed a new plasmid by two-step cloning as shown in Figure 1. At first, we cloned the 78 bp synthetic nirB promoter in a pKK223 derivative plasmid and then the LTB gene (375 bp) was cloned under the control of this promoter and the final plasmid was named pFSnirB78 -23LTB. The second codon of the native LTB gene was changed from AAT to GAT, which altered Asn to Asp in the second amino acid of the LTB signal peptide. The plasmid was transformed into E. coli DH5 in order to measure promotor activity following restriction analysis of the plasmid.

Bottom Line: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes.In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins.Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.

View Article: PubMed Central - PubMed

Affiliation: Semnan Biotechnology Research Center, Semnan University of Medical Sciences, Semnan, IR Iran.

ABSTRACT

Background: Anaerobic-inducible promoters are alternatives of chemical-inducible promoters for expression of recombinant proteins especially in conditions where chemical induction is not possible or anaerobic conditions are preferable. The nirB promoter is the promoter of the first gene of nir operon in Escherichia coli, which encodes NADH-dependent nitrite reductase. This promoter is naturally induced under anaerobic conditions and upregulated by nitrite and nitrate.

Objectives: The current study was carried out to construct a synthetic nirB promoter that does not respond to chemical inducers (nitrite or nitrate), but instead responds to anaerobic induction. For this purpose, a new plasmid was constructed (pFSnirB78-23LTB), which contains a synthetic nirB promoter. The activity of this plasmid was evaluated in E. coli under both aerobic and anaerobic conditions and in response to chemical inducers, nitrite and nitrate.

Materials and methods: A synthetic nirB promoter was firstly cloned into a pKK223 derivative plasmid and then the heat labile toxin B subunit gene (LTB) of entrotoxigenic E. coli was cloned under the control of this promoter. The inducibility of this plasmid in E. coli was measured under anaerobic conditions in the presence or absence of nitrite or nitrate by ganglioside GM1 ELISA.

Results: Our data showed that this promoter is strongly induced under anaerobic conditions while it showed much lower activity (11%) under aerobic conditions. In contrast to the native promoter, this promoter was not induced by chemical inducers, nitrite or nitrate.

Conclusions: This study showed that the recombinant protein produced under the control of synthetic nirB promoter has critical characteristics such as pentamer formation, receptor recognition ability and conservation of antigenic epitopes. In addition, the data showed anaerobiosis and chemical inducers had no adverse effects on recombinant proteins. Based on the results, this synthetic promoter is suitable for use in live delivery vaccines or drug systems and for production of recombinant proteins especially oxygen sensitive proteins.

No MeSH data available.


Related in: MedlinePlus