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.