High frequency plasmid recombination mediated by 28 bp direct repeats

The stability in Escherichia coli of a mammalian expression vector (pCIneo) and its derivative candidate DNA vaccine (pGPV-PV) is described. These multicopy pMB1-type plasmids were found to recombine in several recA E. coli strains due to the presence of two 28 bp direct repeats flanking intervening sequences of 1.6 kb (pCIneo) and 3.2 kb (pGPV-PV). In this recombination event, one of the direct repeats and the intervening sequence were deleted or duplicated, originating monomeric or/and hetero-dimeric plasmid forms, respectively. Additionally, the plasmid rearrangement led to the acquisition of a kanamycin resistance phenotype. Recombination frequencies between 7.8 × 10^-7 and 3.1 × 10^-5 were determined for DH5α and JM109(DE3) strains, respectively. Higher recombination frequencies were found in cells previously grown up to stationary growth phase being the monomeric plasmid form the prevalent one. Real-time PCR quantification revealed the presence of approximately 1.5 × 10⁴ recombined molecules per 2 × 10⁵ cells pre-kanamycin exposure. Under selective pressure of this antibiotic, the number of recombined molecules increased approximately 2,000-fold in a 48-h period replacing the original plasmid form. The high frequency, at which deletion-duplication occurred in the absence of kanamycin selective pressure, should be regarded as a safety concern. This work highlights the impact of mutational hot spots on expression and cloning plasmid vectors and the need to carefully design plasmid vectors.