Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α)

Abstract

Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

“…The construction of the strains BWAA, BW1P, and BW2P, with enhanced uptake for the arabinose inducer (Khlebnikov et al 2001) and deficiency in endA and recA genes that increases the amount and the quality of pDNA (Phue et al 2008;Gonçalves et al 2013), was essential for a higher minicircle production. When ParA was expressed from low-copy number plasmids in the BWAA strain, full recombination of the model parental plasmid pMINI was obtained within 2 h, but no effect of the 5′-UTR was detected (Fig.…”

“…2b) in E. coli BWAA. This strain was derived from E. coli BW27783-a strain improved for arabinose uptake-by knocking out the endA and recA genes to minimize non-specific digestion and recombination of pDNA (Phue et al 2008;Gonçalves et al 2013) and thus assure the maintenance of the supercoiled isoform of minicircle molecules produced. The number of pMMBparA and pMMBpar2A copies per transformed BWAA cell was determined by qPCR to be around 14/cell, a number which is consistent with the 12-13 copies of the pMMB206 reported in the literature (Morales et al 1991).…”

Biomedical application of plasmid DNA in gene therapy: A new challenge for chromatography

“…Typically, DNA vaccine plasmids are manufactured using NIH automatic exempt attenuated E. coli K12 strains such as DH5α , DH5 (Listner et al 2006), DH1 (Cooke et al 2004), JM108, SCS1-L (Singer et al 2009) or DH10B (Lahijani et al 1996. Also, BL21 (an E. coli B strain) has recently been reported as a high yielding plasmid production host (Phue et al 2008). In addition it has been extensively described that for pDNA production auxotrophic strains are suitable for growth in semi-defined media but not for production in minimal media since they require expensive supplementation.…”

“…In an extensive shake flask evaluation of plasmid specific yield and % supercoiling in 17 hosts with three different pUC origins, BL21 (DE3) was the poor producer due to the RecA+ gene product (Yau et al 2008). In contrast, Phue and co-workers reported high yields (1.9 g/L) of predominantly supercoiled plasmid in a 30ºC to 42ºC inducible process (Phue et al 2008). Although these batch fermenta-tions are usually simple and short, they have fundamental disadvantages that result in limited pDNA yields.…”

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

“…To make the StuI site functional, methylation was prevented by transforming target plasmids into the dcm deficient strain Bl21A1 E. coli (Invitrogen, CA). Knowing that Bl21 was recA + /endA + (Phue et al, 2008), we preformed an extra washing step during plasmid preps to remove the excess nucleases (as recommended by the manufacturer). BL21 was not used for long term storage of target plasmids.…”