title: Large-scale plasmid preparation for transient gene expression.
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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Cheng
authors.forename: Xiangming
authors.initials: X
authors.lastname: Sun
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Yi
authors.forename: Yuanxing
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D010957:Plasmids / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism; D014462:Ultrafiltration
chemical_list: D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D012260:Ribonucleases; D005947:Glucose