High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation.

Abstract

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.