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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 (pFS nirB 78-23LTB), 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.

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