质体制备
碱裂解
质粒
溶解
色谱法
超滤(肾)
DNA
化学
大肠杆菌
重组DNA
渗滤
错流过滤
过滤(数学)
大小排阻色谱法
吸附
产量(工程)
分子生物学
生物
dna疫苗
生物化学
材料科学
膜
微滤
PBR322电话
有机化学
基因
数学
冶金
酶
统计
作者
Jason C. Murphy,Michael A. Winters,Sangeetha L. Sagar
出处
期刊:Humana Press eBooks
[Humana Press]
日期:2006-09-20
卷期号:: 351-362
被引量:7
标识
DOI:10.1385/1-59745-168-1:351
摘要
A large-scale approach to the purification of plasmid DNA has been developed that overcomes many of the limitations of current chromatography-based processes. The process consists of a scaleable lysis using recombinant lysozyme and a rapid heating and cooling step followed by a selective precipitation with cetyltrimethylammonium bromide (CTAB). Calcium silicate batch adsorption is then utilized to remove residual genomic DNA, linear plasmid, open circular plasmid, endotoxin, detergents, and proteins. Finally, a concentration and diafiltration step utilizing ultrafiltration and a terminal sterile filtration complete the process. The final product exceeds the requirements for clinical-grade plasmid DNA, and the process has been scaled up to yield an average of 18 ± 4 g (over five lots) of pharmaceutically pure plasmid DNA per 140 L of lysate (from approx 1.3 kg Escherichia coli dry cell weight).
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