Abstract
Large-scale transient gene expression of recombinant protein in mammalian cells requires a great amount of plasmid. An economical method for large-scale plasmid preparation, based on fed-batch fermentation and an improved plasmid extraction process, has been established. Fed-batch growth of E. coli was carried out in 5 l bioreactor by controlling the glucose concentration below 1 g l−1 after the feeding was started. Plasmid yields of 490 and 580 mg l−1 were achieved with two strains of E. coli cells bearing pCEP4-EGFP and pID-EG respectively, representing 24.5- and 26-fold increases over those of the batch culture in shake-flask. To improve the procedure for large-scale preparation of plasmid DNA, addition of RNase to resuspension buffer and ultrafiltration of clarified lysate were adopted, and the quality of the resultant plasmid was comparable to that of commercial kit as disclosed in the small-scale transient transfection. This plasmid production process has great potential in the large scale transient gene expression which needs a large quantity of plasmid DNA.
Similar content being viewed by others
References
Balan S, Murphy J, Galaev I, Kumar A, Fox GE, Mattiasson B, Willson RC (2003) Metal chelate affinity precipitation of RNA and purification of plasmid DNA. Biotechnol Lett 25:1111–1116
Baldi L, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, Derow E, Wurm FM (2005) Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog 21:148–153
Ferreira GNM, Cabral J, Prazeres DMF (1999) Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications. Biotechnol Prog 15:725–731
Girard P, Derouazi M, Baumgartner G, Bourgeois M, Jordan M, Jacko B, Wurm FW (2002) 100-liter transient transfection. Cytotechnology 38:15–21
Kahn DW, Butler MD, Cohen DL, Gordon M, Kahn JW, Winkler ME (2000) Purification of plasmid DNA by tangential flow filtration. Biotechnol Bioeng 69:101–106
Kendall D, Booth AJ, Levy MS, Lye GJ (2001) Separation of supercoiled and open-circular plasmid DNA by liquid-liquid counter-current chromatography. Biotechnol Lett 23:613–619
Lander RJ, Winters MA, Meacle FJ, Buckland BC, Lee AL (2002) Fractional precipitation of plasmid DNA from lysate by CTAB. Biotechnol Bioeng 79:776–784
Muller N, Derouazi M, van Tilborgh F, Wulhfard S, Hacker DL, Jordan M, Wurm FW (2007) Scalable transient gene expression in Chinese hamster ovary cells in instrumented and non-instrumented cultivation systems. Biotechnol Lett 29:703–711
Prazeres DMF, Schluep T, Cooney C (1998) Preparative purification of supercoiled plasmid DNA using anion-exchange chromatography. J Chromatogr A 806:31–45
Rozkov A, Larsson B, Gillström S, Björnestedt R, Schmidt SR (2008) Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture. Biotechnol Bioeng 99:557–566
Tait AS, Brown CJ, Galbraith DJ, Hines MJ, Hoare M, Birch JR, James DC (2004) Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents. Biotechnol Bioeng 88:707–721
Wright JL, Jordan M, Wurm FM (2001) Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection. Cytotechnology 35:165–173
Wurm F, Bernard A (1999) Large-scale transient expression in mammalian cells for recombinant protein production. Curr Opin Biotechnol 10:156–159
Acknowledgements
This work was supported by grants from the Open Project Funding of State Key Laboratory of Bioreactor Engineering of China and National Special Project of Key Technology of China (2009ZX09503-013-2, 2009ZX09102-249).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Cheng, L., Sun, X., Yi, X. et al. Large-scale plasmid preparation for transient gene expression. Biotechnol Lett 33, 1559–1564 (2011). https://doi.org/10.1007/s10529-011-0612-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-011-0612-x