Patent
General methods and strains of bacteria are described that dramatically simplify and streamline plasmid DNA production. In one preferred embodiment, endolysin mediated plasmid extraction combined with flocculation mediated removal of cell debris and host nucleic acids achieves increased yield and purity with simplified downstream purification and reduced waste streams, thus reducing production costs.
1. A method for increasing production yield of a covalently closed, super-coiled plasmid DNA vector that is non-repetitive and does not contain direct repeats or long terminal repeats, comprising the steps of:
a. transforming a plasmid DNA vector that is non-repetitive and does not contain direct repeats or long terminal repeats into bacterial cells rendered competent for transformation; and
b. growing resultant transformed bacterial cells at a reduced temperature during colony isolation and colony amplification steps in order to create a cryopreserved seed stock for plasmid DNA vector production, the reduced temperature being approximately 20-33° C.;
whereby growth at said reduced temperature prior to cryopreservation improves said plasmid DNA vector yield in subsequent shake flask and or fermentation culture above yield that is achieved when growing the transformed bacterial cells at a temperature approximately 33-42° C. during colony isolation and colony amplification steps.
2. The method of claim 1 wherein said bacterial cells are Escherichia coli cells.
3. The method of claim 1 wherein said plasmid DNA vector that is non-repetitive and does not contain direct repeats or long terminal repeats is a DNA vaccine plasmid encoding an H1 or H3 serotype influenza hemagglutinin gene.