State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
Biotechnol Lett. 2011 Aug;33(8):1559-64. doi: 10.1007/s10529-011-0612-x. Epub 2011 Apr 8.
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.
大规模瞬时基因表达重组蛋白在哺乳动物细胞中需要大量的质粒。基于分批发酵和改进的质粒提取工艺，建立了一种经济的大规模质粒制备方法。通过在开始进料后将葡萄糖浓度控制在 1g/L 以下，在 5L 生物反应器中进行大肠杆菌的分批补料生长。带有 pCEP4-EGFP 和 pID-EG 的两种大肠杆菌细胞的质粒产量分别达到了 490 和 580mg/L，分别比摇瓶分批培养提高了 24.5 倍和 26 倍。为了改进大规模制备质粒 DNA 的程序，在重悬缓冲液中添加了 RNase，并对澄清的裂解物进行了超滤，得到的质粒的质量与小规模瞬时转染中商业试剂盒相当。该质粒生产工艺在需要大量质粒 DNA 的大规模瞬时基因表达中具有很大的潜力。
