High-Cell-Density Culture to Produce Plasmid DNA for Gene Therapy in E. coli

How to produce Plasmid DNA in a high-cell-density culture.


The recombinant host Escherichia coli DH10B bearing the plasmid pIDKE2 was grown under fed batch conditions, and the effects of different medium components on plasmid yield and cell mass were evaluated at a 5-L fermentation scale. Results showed that glucose was the optimal carbon source at 265 g/L. After testing different levels of nitrogen, a defined complex medium was formulated for optimal plasmid production, capable of producing up to 0.44 mg plasmid DNA per g dry cell weight.

Interest in gene therapy has grown considerably over the last decade because of its promise as a treatment for the prevention, treatment, and cure of diseases such as cancer and acquired inmudeficiency syndrome (AIDS).1,2 It has been shown that naked DNA injected into muscle tissue is expressed in vivo and that the introduction of immunogenic sequences can result in animal vaccination against the encoded peptide.3,4 In general, DNA-based vaccines are considered very safe, in part because of the lack of genetic integration, and also because of the absence of a specific immune response to the plasmid itself.5 This safety makes the use of DNA vaccines very attractive.6 In addition, unlike live attenuated vaccines, plasmid DNA (pDNA) vaccines do not carry the hypothetical risk of reverting to a viable state and causing illness.7

Gene therapy treatments require considerable amounts of pDNA, which must be homogeneous with respect to its structural form and DNA sequence. Certain growth conditions can lead to significant amounts of non-supercoiled pDNA forms. This means that the homogeneity and quality of the final pDNA product will be a function of interactions between the host, the plasmid, and the growth environment. Therefore, the choice of fermentation protocol will be critical to minimize process contaminants that need to be removed during downstream processing.8

A further feature of fermentation processes for large-scale plasmid production is the performance of high-density fermentations to obtain large amounts of biomass. Experimental work on the composition of bacterial growth media has demonstrated that fermentation conditions and growth media strongly influence the yield and quality of plasmids produced in E. coli cells.9

In this work, the medium composition of a high-cell-density culture for the production of plasmid pIDKE2 in a recombinant E. coli DH10B system was studied. The results provide a defined medium for pDNA production.


Strain and Plasmid

The plasmid used in this work was pIDKE2 with a kanamycin-resistant marker; it was transfected into E. coli DH10B using standard methods.1 The E. coli was then grown on several LB agar plates supplemented with 50 μg kanamycin/mL.

Culture Media

The basic LB medium for seed cultivation contained 10 g tryptone/L, 5 g yeast extract/L, and 10 g NaCl/L, supplemented with 50 μg kanamycin/mL. A fed-batch fermentation was carried out in a 5-L bioreactor with a working volume of 4 L, in a complex medium containing 5 g glucose/L, 1.4 g KH2PO4/L, 8.6 g (NH4)2SO4/L, 1 g MgSO47H2O/L, 10 g yeast extract/L, 1 mL trace metal solution 1,000 X/L, and 50 μg kanamycin/mL. The trace metal solution 1X was prepared as follows: 2.29 mg AlCl3·6H2O/L, 1.6 mg (CoCl2·6H2O)/L, 41.21 mg H3BO3/L, 4.98 mg (MnSO4·H2O)/L, 0.73 mg (Na2MoO4·2H2O)/L, 13.7 mg (CuSO4·H2O)/L. Using a peristaltic pump, feed medium (265 g glucose/L and 50 g yeast extract/L) was added to the fed batch cultures at a constant flow rate of 0.7 mL/min when the pH had risen, because that was a signal that the initial carbon source had been depleted.

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