Characterization of a Novel Overexpression System in Haloferax volcanii

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A novel inducible protein expression system has been developed in the halophilic archaea, Haloferax volcanii (Hvo.) The expression system is based on the histidine utilization (Hut) operon and can be induced by histidine or urocanate, a histidine metabolite. This study characterizes the 88 base promoter region between the gene that encodes a newly discovered Hut regulatory protein (Hut R) and the first gene in the Hut operon, Hut U. Plasmids have been constructed that contain the gene for Hut R, the promoter region, the first three codons of Hut U and the gene for L7ae, an RNA binding protein, used as an expression reporter (see figure). The plasmid containing the wildtype promoter yields a high level of inducible gene expression when transformed into Hvo. Two specific mutations have been constructed in the promoter near the Hut U start sequence, deleting either 5 base (Del 1) or 20 bases (Del 2) immediately upstream of the Hut U start codon. These mutants yield decreased gene expression as measured by Northern and Western analysis. The new system appears to be generally useful for overexpression of halophilic proteins and has the potential for protein overexpression from a variety of halophilic organisms. This work was supported by Capital University.