Patent Evaluation: A Stable Expression Vector for E. coli

Summary

Novelty: A stable vector for E. coli is described which does not have a constitutive resistance gene and is segregationally stable because of this resistance. The plasmid also contains a cer sequence to give added stability by resolving plasmid multimers. This will be of particular use in the large scale fermentation of recombinant proteins as it is not necessary to continuously add antibiotics in this system.

Biology: The plasmids are based on the plasmid pAT153 with the normal antibiotic resistance genes removed and replaced by the tetA and tetR genes from RP4. These two genes allow inducible resistance to tetracycline and are only expressed when tetracycline is present in the medium. The cer sequence from ColE1 was also placed in the vectors. Heterologous genes such as G-CSF, Ricin and alpha 2 interferon were cloned into such vectors under the control of promoters such as lambda pL, trp and T7.

Chemistry: The stability was 100% after 150 generations under non-selective conditions.