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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

Comparison of Sequences of Intact Native nirB Promoter (GenBank gi:42120), pTETnir15 (14) and pFSnirB78-23 (this study)In pFSnirB78-23 plasmid the FNR region is visualized by underlined capital letters, ribosome-binding site (RBS) is shown by bold and -10 region by italic capital letters.
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fig12085: Comparison of Sequences of Intact Native nirB Promoter (GenBank gi:42120), pTETnir15 (14) and pFSnirB78-23 (this study)In pFSnirB78-23 plasmid the FNR region is visualized by underlined capital letters, ribosome-binding site (RBS) is shown by bold and -10 region by italic capital letters.

Mentions: Intact native nirB promoter has also been used for expression of foreign genes in Salmonella live vaccine strains and has been shown to be an efficient system for immunization (17). With the aim of dissociation of chemical induciblity from anaerobiosis, we constructed an engineered synthetic nirB promoter, and evaluated the expression of LTB gene under anaerobic conditions, and determined the regulatory effects of nitrite and nitrate as chemical inducers on the activity of this promoter. We considered some critical regulatory regions on this engineered promoter. Firstly, we considered a hexamer sequence, TAAGGT at -10 position that is necessary for anaerobic activation. It has been shown that mutation in this region may result in loss of activity of the promoter (8). Secondly, a ribosome binding site (RBS) sequence was examined for control of translation. Thirdly, we evaluated the FNR global regulatory protein-binding site at -30 and -52, related to transcription start position. FNR protein encoded by the fnr gene regulates protein expression under anaerobic conditions via activation of transcription initiation of some anaerobic promoters such as nirB (7). We compared our nirB promoter with the intact native promoter and a synthetic promoter (pTETnir15) reported by Oxer et al. (14), as shown in Figure 2. There were some differences in the sequences that explain the probable variation between their activities.


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

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

Comparison of Sequences of Intact Native nirB Promoter (GenBank gi:42120), pTETnir15 (14) and pFSnirB78-23 (this study)In pFSnirB78-23 plasmid the FNR region is visualized by underlined capital letters, ribosome-binding site (RBS) is shown by bold and -10 region by italic capital letters.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig12085: Comparison of Sequences of Intact Native nirB Promoter (GenBank gi:42120), pTETnir15 (14) and pFSnirB78-23 (this study)In pFSnirB78-23 plasmid the FNR region is visualized by underlined capital letters, ribosome-binding site (RBS) is shown by bold and -10 region by italic capital letters.
Mentions: Intact native nirB promoter has also been used for expression of foreign genes in Salmonella live vaccine strains and has been shown to be an efficient system for immunization (17). With the aim of dissociation of chemical induciblity from anaerobiosis, we constructed an engineered synthetic nirB promoter, and evaluated the expression of LTB gene under anaerobic conditions, and determined the regulatory effects of nitrite and nitrate as chemical inducers on the activity of this promoter. We considered some critical regulatory regions on this engineered promoter. Firstly, we considered a hexamer sequence, TAAGGT at -10 position that is necessary for anaerobic activation. It has been shown that mutation in this region may result in loss of activity of the promoter (8). Secondly, a ribosome binding site (RBS) sequence was examined for control of translation. Thirdly, we evaluated the FNR global regulatory protein-binding site at -30 and -52, related to transcription start position. FNR protein encoded by the fnr gene regulates protein expression under anaerobic conditions via activation of transcription initiation of some anaerobic promoters such as nirB (7). We compared our nirB promoter with the intact native promoter and a synthetic promoter (pTETnir15) reported by Oxer et al. (14), as shown in Figure 2. There were some differences in the sequences that explain the probable variation between their activities.

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