Article

Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5alpha).

Biotechnology Core Laboratory, NIDDK NIH Bethesda, Bldg 14A Room 173, Maryland 20892, USA.
Biotechnology and Bioengineering (Impact Factor: 4.16). 06/2008; 101(4):831-6. DOI: 10.1002/bit.21973
Source: PubMed

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.

3 Bookmarks
 · 
733 Views
  • Source
    -, -; 04/2006
  • [Show abstract] [Hide abstract]
    ABSTRACT: The need for the development of economic high plasmid production in Escherichia coli cultures is emerging, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5α is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described different culture behaviors, either using glucose or glycerol as carbon source, or mixtures of both. From the seventeen parameters obtained after model simplification, the following ten parameters were found to be independent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control.
    Journal of Biotechnology 07/2014; · 2.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼ 141mg/L) than MG1655ΔendAΔrecA (∼ 48mg/L) and DH5α (∼ 40mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200mg/L.
    Journal of Biotechnology 07/2014; · 2.88 Impact Factor

Full-text

Download
690 Downloads
Available from
May 22, 2014