A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA.
There is an increasing interest and need for the development of scaleable process for the preparation of plasmid DNA for vaccines and gene therapy. In this report, we describe a streamline modified process of plasmid extraction based on boiling lysis in order to simplify the operation and process large volumes of Escherichia coli cultures. The bacteria, harvested using a hollow fiber cartridge after fermentation, were treated with lysozyme at 37 degrees C prior to passing through a heat-exchanger coil. Subsequently, the supernatant was separated from lysed bacteria using a 65 microm nylon filter. The employment of a peristaltic pump and two heating coils at constant temperature without the use of centrifugation enabled the process protocol to be constant and controllable. A relatively low lysis temperature of approximately 70-80 degrees C and a buffer modified for the high-density cultures were also optimized for the process. Prior to thermal lysis, a pre-treatment step with the lysozyme for 20 min at 37 degrees C was one of the crucial steps contributing to the high plasmid quantity and quality from batch to batch. After harvesting 17 L of E. coli cultures (OD600 = 50), the plasmid can be extracted within 45 min with this streamline protocol. The plasmid yields are approximately 100mg/L culture, which makes it attractive and promising for the large-scale preparation of plasmid.
State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University, Beijing 100094, China.
Kaichun
K
Zhu
Huali
H
Jin
Yijie
Y
Ma
Zhihui
Z
Ren
Chong
C
Xiao
Zhonghuai
Z
He
Fuchun
F
Zhang
Qinghong
Q
Zhu
Bin
B
Wang
D018929:Cell Culture Techniques / Q000379:methods*; D002458:Cell Fractionation / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics* / Q000302:isolation & purification*; D006358:Hot Temperature; D010957:Plasmids / Q000235:genetics / Q000302:isolation & purification*
D004269:DNA, Bacterial