Abstract
There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.
Highlights
The past few decades have seen a rise in the focus of plasmid DNA for biotherapeutic production, with applications including vaccines and gene therapy
We investigate the impact of the bacteriophage Mu strong gyrase binding site sequence engineered into a 6.8 kb plasmid and amplified in four different E. coli strains which have been selected based on past studies, as well as to examine the effect of the potential increase in supercoiling on the integrity of the plasmid during downstream processing
A significantly higher plasmid yield was observed for SGS-containing plasmid pSVβ-gal398 as compared with the parent plasmid pSVβ-gal
Summary
The past few decades have seen a rise in the focus of plasmid DNA for biotherapeutic production, with applications including vaccines and gene therapy. It has been reported that by achieving a high cell density culture, the plasmid yield is improved as plasmid production is directly linked to the biomass yield of a culture [10]. Some of these reports have achieved high biomass yield by engineering the cell using gene knockout or gene overexpression. Examples include the knockout of the pts gene responsible for glucose transport and replacement with galp+ which codes for permease [11,12], modification of pentose phosphate pathway [13], and knocking out the pyruvate kinase gene [14]
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