Large-scale plasmid preparation for transient gene expression |
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Authors: | Lu Cheng Xiangming Sun Xiaoping Yi Yuanxing Zhang |
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Institution: | (1) State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China;(2) Shanghai Kexin Biotech Co. Ltd, Shanghai, 201203, China; |
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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. |
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