High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation.
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.
Lahijani
R
R
Vical, Incorporated, San Diego, CA 92121, USA.
Hulley
G
G
Soriano
G
G
Horn
N A
NA
Marquet
M
M
D004269:DNA, Bacterial / Q000096:biosynthesis*; D005285:Fermentation; D015316:Genetic Therapy; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D017354:Point Mutation; D013696:Temperature
D004269:DNA, Bacterial