In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-β-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction.
Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an α-complementation assay, in which the α-peptide of the lacZ (β-galactosidase) gene is inserted in XL-10 E. coli cells. With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the original solo expression (pgMAX) system. Furthermore, recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP) with DsRed in arabinose unit and EGFP in lac unit showed expression of each fluorescent proteins, although the expression levels were limited. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses.