Recombinant protein production with minimal-antibiotic-resistance vectors

Commonly used expression vectors direct the synthesis of antibiotic-resistance proteins at unnecessarily high levels that complicate purification of the desired recombinant product. To overcome this problem, the promoter of the kanamycin-resistance gene (kanR) was altered by site-specific mutagenesis. As a result, synthesis of KanR protein was greatly reduced to the minimum required for host selection. At the same time, recombinant protein production was increased up to two-fold. Since the mutations did not alter any coding sequences and had no effect on plasmid copy number, the results suggest that plasmid-coded protein production can be limited, at least in part, by other genes expressed from the same plasmid. Because of the dependence of protein synthesis on gene dosage, an important aspect of minimizing antibiotic resistance is continuous selection for cells harboring the maximum vector-copy number, thus ensuring maximal product synthesis.