Method for increased yields of plasmid DNA
J. Gowrishankar et al.)
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.
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.
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.
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:
Gowrishankar, J., and Harinarayanan, R. 2004. Why is transcription coupled to translation in bacteria? Mol. Microbiol.