文献类型：期刊文献

英文题名：Large-scale plasmid preparation for transient gene expression

作者：Cheng, Lu[1];Sun, Xiangming[2];Yi, Xiaoping[1];Zhang, Yuanxing[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Shanghai Kexin Biotech Co Ltd, Shanghai 201203, Peoples R China

年份：2011

卷号：33

期号：8

起止页码：1559

外文期刊名：BIOTECHNOLOGY LETTERS

收录：;EI(收录号：20112814147529);WOS:【SCI-EXPANDED(收录号:WOS:000293752000008)】；

基金：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).

语种：英文

外文关键词：CHO cells; Fed-batch; Plasmid DNA; RNase; Transient transfection; Ultrafiltration

摘要：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.