Method for increased yields of plasmid DNA

(Inventors: J. Gowrishankar et al.) 

Brief description:   

The invention exploits a novel phenotype identified by the inventors of E. coli strains defective in either of two factors Rho or NusG involved in transcription termination, whereby the strains are killed upon transformation with ColE1-like plasmids such as pUC19 and pACYC184 consequent to uncontrolled (“runaway”) replication of the plasmids in the mutant strains. The invention describes processes for obtaining increased yields of plasmid DNA in which the plasmid is introduced into a strain carrying both a chromosomal rho or nusG mutation and the cognate wild-type gene on a conditional replicon vector (eg., IPTG-dependent for replication), and then subculturing into medium under restrictive conditions (eg., without IPTG) so that the wild-type gene is lost and the plasmid undergoes uncontrolled replication. The yield of plasmid DNA is typically 10-fold or more higher from the mutant strain than it is from control strains.  


Plasmid DNA has become a pharmaceutical product in its own right with the advent in recent years of prophylactic and therapeutic DNA vaccines for a variety of diseases including influenza, SARS, anthrax, rabies, Alzheimer’s, and cancer please click here for an illustrative list of high-profile papers describing these applications. 

      In the preparation of DNA vaccines, the major bottleneck is in the yield of plasmid DNA per litre of culture – the typical yield is about 4 to 5 mg/L, and the vaccination protocol usually is three doses of 100 μg each per individual. Hence, processes that lead to increased yields of plasmid DNA are expected to be very useful in the art. 

Research publications:

Harinarayanan, R., and Gowrishankar, J. 2003. M Host factor titration by chromosomal R-loops as a mechanism for
           runaway plasmid replication in transcription termination-defective mutants of Escherichia coli. J. Mol. Biol. 332:
           31-46. [PDF] 

Gowrishankar, J., and Harinarayanan, R. 2004. Why is transcription coupled to translation in bacteria? Mol. Microbiol.
           54: 598-603.[PDF]

Patents filed: 

[PCT]     [Europe]     [US]     [US-Divisional]     [Japan] 

Search Reports:    

[International]     [Europe]