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. - BioPharm International

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

Effects of Nitrogen Source on Plasmid Production

The choice of nitrogen source and the determination of its concentration are critical to the optimization of plasmid production in recombinant cell fermentation. The bacterial requirements for nitrogen can be satisfied by inorganic or organic sources. In our work, yeast extract was used and the effects of different levels in the feed were tested.


Figure 4. Effects of yeast extract concentration on plasmid production and cell growth in a 5-L fed batch fermentation process
Results (Figure 4) showed that the cell mass increased up to 28 g dry cell weight/L when the concentration of yeast extract was increased from 20 g/L to 50 g/L and the highest specific plasmid productivity (0.45 mg plasmid DNA g/L dry cell weight) was achieved. Therefore, from the viewpoint of the development of high-cell-density culture, an inorganic supply of nitrogen from complex components such as yeast extract was essential because it was more effective in supporting high plasmid yield.

CONCLUSION

With this cell culture procedure, larger amounts of the plasmid pIDKE2 were obtained in DH10B cells. The results from this study may be beneficial for the development of techniques for fed batch fermentation of E. coli cells and for the efficient production of plasmid DNA for therapeutic use in humans.

Odalys Ruiz Hernández is the principal researcher, Mariela Pérez de la Iglesia, Saily Martínez Gómez, Karelia Macias Cosme, Michel Díaz Martinez, and Yanay Proenza Jimenez are researchers in the fermentation department and Jorge Valdés Hernández is the department head. Miladys Limonta Fernández is the principal researcher in the purification department. Marta Pupo Peña is a researcher and Dinorah Torres Idahody, PhD, is the department head, both in the analytical development department, and Eduardo Martínez, PhD, is the head of the development division, all at the Center for Genetic Engineering and Biotechnology, Havana, Cuba,
+537.271.6022, ext: 5250.


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