Appl Microbiol Biotechnol (2007) 75:12171223 DOI 10.

1007/s00253-007-0925-9

METHODS

An automated process to extract plasmid DNA by alkaline lysis

Xiaolin Li & Huali Jin & Zhifang Wu & Simon Rayner & Jiangmei Yin & Yang Yu & Wenjuan Zhang & Zhonghuai He & Chen Wang & Bin Wang

Received: 2 January 2007 / Revised: 2 March 2007 / Accepted: 4 March 2007 / Published online: 27 March 2007 # Springer-Verlag 2007

Abstract With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture. Keywords Plasmid . Large-scale . Alkaline lysis . Automated process

Introduction Rapid advances in the fields of DNA vaccines and gene therapy (Friedmann 1997; Liljeqvist and Stahl 1999) have produced increased demands for large quantities of recombinant plasmid DNA. The problem is further exacerbated because the efficiency of plasmid-based delivery to cells is relatively lowonly 0.1% of plasmids presented to the cells can reach the nucleus and be expressed (Crystal 1995; Ferreira et al. 2000; Levy et al. 2000). Thus, a scaleable process for the automated preparation of plasmid DNA would be of great benefit to researchers and is a problem that has been the focus of previous studies by biochemical engineers (Eastman and Durland 1998; Levy et al. 2000). Broadly speaking, a process for manufacturing plasmid DNA includes the following steps: plasmid construction, cell growth and extraction, and purification of the plasmid (Ferreira et al. 2000; Prather et al. 2003). For extraction and purification, the most critical part of the process is probably cell breakage (Prazeres et al. 1999). Several ways can be used to break the cells. The most popular method is based on alkaline lysis of the cell (Lahijani et al. 1996). The principal drawback with this method, with a view to automating the process, is that it includes multiple centrifugation steps. The first step separates the bacteria from the cultured media before lysis; the second step separates the plasmid from the cell debris after lysis, and the third step separates the plasmid from RNA after resuspension (Kelly and Hatton 1991; Prazeres et al. 1998). These steps cannot be easily incorporated into an automated process and impede scaling up the alkaline lysis method for large-scale preparation of plasmid DNA (Theodossiou et al. 1997). An alternative method for cell breakage is boiling lysis (Sambrook et al. 1989). However, in this approach, the yield and purity of the plasmid is inconsistent and the complexities

X. Li : H. Jin : Z. Wu : J. Yin : Y. Yu : W. Zhang : Z. He : C. Wang : B. Wang (*) State Key Laboratory for Agro-Biotechnology, Key Laboratory of Agro-Microbial Resources and Applications of MOA, College of Biological Science, China Agricultural University, Beijing 100094, China e-mail: bwang3@cau.edu.cn

Present address: S. Rayner Wuhan Institute of Virology, Chinese Academy of Science, Wuhan, Hubei

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of the method make it undesirable even for research scale preparation (Wang et al. 1995). Typically, in a large-scale extraction, high-pressure homogenizers are used. While this process is effective at disrupting cells in an automated fashion, the severity of the shearing force experienced by the cells can result in the degradation of the plasmid DNA (Clemson and Kelly 2003). More recently, Zhu et al. (2005) successfully developed and optimized an automated process based on the thermal lysis protocol. The process was modified from the conventional boiling lysis protocol and made plasmids in a continuous and scalable manner. However, a centrifugation step is still required after lysis. We sought to develop a process based on the traditional alkaline lysis procedure, but with the incorporations of such modifications, as to allow a more automated process. Specifically, we have developed a novel design comprising automated control of reagent flow into the system and replacement of all of the centrifugation steps by use of filters and hollow membranes. The resulting automated process provided a scalable, faster, and more efficient method for plasmid preparation that could better meet the needs of various clinical and research applications.

Na2HPO4, 1.2 g/l (NH4)2SO4, 0.2 g/l NH4Cl) with 50 g/ml of kanamycin or ampicillin. The temperature was set at 37C, and the dissolved oxygen (DO) value was maintained around 30% with the program set to cascade control with a speed-shifted agitation. The pH was adjusted to 6.87.0 and was controlled by pumping in ammonia solution (30%) as the alkali and 4 M of hydrogen chloride (HCl) as the acid. The filtered inflow air was set at the ratio of 1:1, and 400 ml of concentrated nutrient (71 g/l yeast extract, 71 g/l pepton, 170 g/l glycerol, and 5.7 g/l MgSO4) was fed to the fermentor at the rate of 20 ml/h. Four milliliters of culture was collected on each hour during fermentation, and the optical density at 600 nm was measured. Harvesting of bacteria Bacteria cells from the fermentation were harvested using a hollow, 0.2-m pore-sized hollow-fiber membrane module (Tianjin Motian Membrane Engineering and Technology, Tianjin, China) and rinsed once with solution I (50 mM Glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) within the hollow fiber system and was stored at 20C until use. Automated alkaline lysis preparation

Materials and methods Reagents DNA Markers and restriction enzymes were purchased from TaKaRa Biotechnology (Japan). Yeast extract and tryptone were purchased from Oxford (Japan). The ampicillin and kanamycin were purchased from Amresco (USA). The chemicals were purchased from Beijing Chemical Reagents (China). The plasmid DNA, pcDNA3VP1, encoded a capsid protein VP1 gene (639 bp) of the foot and mouth disease virus (FMDV; Jin et al. 2004). The Escherichia coli strains DH5, pcDNA3, pVAX1, and pEGFP-N3 were purchased from Invitrogen. The BHK-21 cells were maintained in Dulbeccos modified Eagles medium 10 (DMEM-10) medium at the lab. Fermentation The plasmid was transformed into DH5 and propagated in LuriaBertani (LB) medium (Sambrook et al. 1989). A single colony of bacteria carrying the plasmid was picked out and inoculated into 50 ml of LB culture containing 50 g/ml kanamycin at 37C and shaken overnight. Subsequently, 40 ml of the culture was inoculated into the 7-l fermentor vessel (Bio-flo 110, NBS, New Browswick, NJ, USA) containing 4 l of semi-synthesized medium (5 g/l trypton, 5 g/l yeast extract, 4 g/l KH2PO4, 4 g/l K2HPO4, 7 g/l

The harvested bacteria were resuspended in solution I (50 mM Glucose, 25 mM Tris-HCl, 10 mM EDTA, and pH 8.0) until the OD value at 600 nm reached 100. The diluted bacteria, 0.4 M of NaOH, 2% of sodium dodecyl sulfate (SDS), and solution III were pumped into two cells for mixing through different silica tubes (with an inner diameter of 3 mm) at a speed of 3.0 cm/s (Fig. 1). In cell A, bacteria from pump 2 (P2) and tube 1 (T1) were mixed with 0.4 M of NaOH from pump P3 and tube T2, and 2% SDS from pump P4 and tube T3. The shape and size of cell A is critical for the complete lysis. In cell B, the lysed bacteria were neutralized with solution III (3 M potassium acetate, 5 M acetic acid, and pH 4.8) from C5 with constant shaking (at 75 Hz). To filter out the debris and insoluble sludge, the final mixture was passed through a series of stacked filters N that comprised four sequential nylon filters with pore sizes of 375, 186, 122, and 70 m. The liquid was collected in container C6, containing 100% ethanol at two times the combined volume of containers C2+C3+C4+C5. The precipitated plasmid was then collected through a nylon filter of 70-m pore size. Manual alkaline lysis preparation The manual alkaline preparation was done according to a previously documented protocol (Sambrook et al. 1989) with a minor modification. The harvested bacteria above were resuspended with solution I until the OD value at

Appl Microbiol Biotechnol (2007) 75:12171223 Fig. 1 Schematic representation of the automated process for alkaline lysis. The device comprises the following apparatus: F fermenter; M hollow fiber membrane module; P1, P2, P3, P4, and P5 peristaltic pumps of which P2, P3, P4, and P5 operate at the same speed; A a mixing cell for bacteria and solution II; B a mixing cell for solution III and the lysed bacteria; C1 waste reservoir; C2 reservoir for bacteria in solution I; C3 0.4-M NaOH reservoir; C4 2% SDS reservoir; C5 reservoir for solution III; C6 100% ethanol reservoir; N four nylon filters of 375, 186, 122, 70 m pore sizes. Tubes T1, T2, T3, and T4 transfer different solutions between reservoirs and mixing cells

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600 nm reached 100. The remainder of the protocol was then performed according to the documentation (Sambrook et al. 1989). Plasmid isolation and purification The pellet of the plasmid DNA was resuspended with an appropriate volume of TrisEDTA (TE) and mixed with the same volume of 5 M LiCl. After centrifugation at 12,000 rpm for 10 min, the supernatant was transferred into a clean flask and mixed with 0.1 volume of 3 M NaAc (pH 5.2) and 0.6 volume of isopropanol at room temperature for 20 min before a second centrifugation at 12,000 rpm for 10 min. The precipitation was resuspended with an appropriate volume of TE. After RNA removal by 100 ug/ml of RNase A digestion at 37C for 2 h, the plasmid was purified using a Qiagen-tip according to the

manufacturer s instructions (Qiagen, Germany). The purified plasmid DNA was then dissolved in TE buffer for further use. Agarose gel electrophoresis Plasmid samples were run on a 0.7% agarose gel containing 0.05 g/ml of ethidium bromide. The gel was viewed under 300-nm UV light and recorded by a digital camera (Olympus, Japan). Plasmid measurement The plasmid was serial diluted and run on a 0.7% agarose gel along with a standard DNA marker (TaKaRa). All bands were visualized under UV light after staining by ethidium bromide and recorded by digital camera (Olympus, Japan). Each band

Fig. 2 Effects of cell concentration. Bacteria were resuspended in solution I; the OD600 nm values were adjusted to 50, 100, 150, 200, 250, 300, and 350 by dilution of solution I. a After the cell was lysed, plasmid was extracted and RNA was removed. Five microliters of each sample was loaded and run on a 0.7% agarose gel to determine its purity and yield. M

The DNA marker DL15000; C plasmid prepared by manual alkaline lysis; lanes 17 OD600 nm 50, 100, 150, 200, 250, 300, and 350. OC Plasmid in open circle form; SC plasmid in supercoil form. b The intensity of each SC band was analyzed by Quantity One software. Data was representative of three independent experiments

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Fig. 3 Effects of the mixing order of the solutions. Different combinations for the solution mixing order were tested by re-plumbing the system (i.e., rearranging the connectivity of tubes T1, T2, and T3 in Fig. 1). a Plasmid was extracted and RNA was removed. Five microliters of each sample was loaded and run on a 0.7% agarose gel to determine its purity and yield. M The DNA marker, DL15000; C plasmid prepared by manual alkaline lysis; lane 1 0.4-M NaOH and 2% SDS mixed first, then mixed with bacteria; lane 2 bacteria and 0.4-M NaOH mixed first, then mixed with 2% SDS; lane 3 bacteria and 2% SDS mixed first, then mixed with 0.4-M NaOH. b The intensity of each SC band was analyzed by Quantity One software. Data was representative of three independent experiments

lysing, the concentrated bacteria was manually adjusted to 100 of OD600 nm by solution I and collected in reservoir C2. The solution was then pumped into mixing cell A. The cell is of circular cross-section (2-cm diameter) and elliptically shaped along the longitudinal axis (8 cm long), similar to a rugby ball, which also receives inlet streams of 0.4 M NaOH (P3, T2) and 2% SDS (via P4, T3). As a consequence of the lysing process, the resulting solution was of high alkalinity and viscosity and was passed to a second mixing cell B where it was mixed with solution III and neutralized. To overcome mixing problems due to the viscosity, mixing cell B was attached to a horizontal shaker (Lab-Line Instruments) and shaken at 75 Hz. After shaking and mixing, most proteins and chromosomal DNAs that formed white precipitations were removed by passing the solution through filter N comprising four sequential nylon membranes of progressively smaller pore size (375, 186, 122, and 70 m). Liquid passing though the filter was mixed with the ethanol in container C6, subjected to a gentle stirring, and the crude plasmid DNA was precipitated. The precipitated plasmid was collected by a 70-m nylon filter, rinsed twice with 70% ethanol, and dried in air. The dried plasmid was resuspended in TE solution and subjected to further purification. Optimization of the system There are a number of parameters that can affect the quality and yield of the plasmid DNA extracted. Therefore, to

was analyzed for its quantity by comparison with the intensity of a known amount of DNA marker using the Quantity One software package (Bio-Rad, CA, USA). Transfection We mixed 10 g plasmid pEGFP-N3 that was previously purified with lipofectamine 2000 reagent (Invitrogen, USA) and transfected the plasmid into the cell strain BHK21 according to the manufacturer s instructions. The cells were cultured in DMEM 10% fetal bovine serum for 48 h. The level of expression was measured by the intensity of fluorescence in the fluorescent-activated cell sorting (FACS; BD Biosciences, USA) to determine the transfection efficiency.

Results Construction and operation of a flow-through device A schematic of the apparatus is shown in Fig. 1. The apparatus consists of a fermenter, a series of reservoirs for the systematic introduction of each of the reagents at the appropriate point in the process, and two mixing cells. The flow rates for each reagent are controllable via a corresponding set of peristaltic pumps. The solution from the fermenter was first passed through a hollow fiber column to separate and concentrate the bacteria. Before

Fig. 4 Effect of the mixing cell for bacteria and solution II. a After the plasmid was extracted from the lysed bacteria with or without the mixing cell A, RNA was removed, and 5 l of each sample was loaded and run on a 0.7% agarose gel to determine its purity and yield. M The DNA marker DL15000; C plasmid prepared by manual alkaline lysis; lane 1 with mixing cell A; lane 2 without mixing cell A. b The intensity of each SC band was analyzed by Quantity One software. Data was representative of three independent experiments

Appl Microbiol Biotechnol (2007) 75:12171223 Fig. 5 Effect of flow rate. a After the plasmid was extracted from the lysed bacteria at different flow rates, RNA was removed, and 5 l of each sample was loaded and run on a 0.7% agarose gel to determine its purity and yield. M The DNA marker, DL15000; C plasmid prepared by manual alkaline lysis; lanes 16 plasmids prepared at flow rates of 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 cm/s, respectively. b The intensity of each SC band was analyzed by Quantity One software. Data was representative of four independent experiments

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determine the experimental conditions to optimize the system, we examined the following parameters. Effects of cell concentrations The cell concentration of the bacteria in solution I can affect the efficiency of the process. To examine this effect, concentrated bacteria from the hollow fiber column were collected and manually diluted into 50, 100, 150, 200, 250, 300 and 350 of OD600 nm, respectively, by mixing with solution I. Fifty milliliters of each concentration was tested by flowing through the system at a rate of 2.5 cm/s through 3-mm diameter silica tubing. After collection from C6, the samples were centrifuged, resuspended in TE buffer, and treated with RNase to remove RNA. The quantity and quality of plasmid was examined by running on a 0.7% agarose gel. The results showed that the best yield was obtained from the bacteria with a concentration of 100 OD600 nm (Fig. 2). This cell concentration was used in all subsequent tests.

Effects of the mixing order for solutions We next investigated the effect of the mixing order for bacteria, NaOH, and SDS to determine whether this plays an important role in the overall efficiency of the process. To find the optimal sequence for the mixing of NaOH, SDS, and bacteria, we tested different combinations for the solution mixing order by re-plumbing the system (i.e., rearranging the connectivity of tubes T1, T2, and T3 in Fig. 1) in the following ways: (1) 0.4 M NaOH and 2%SDS was mixed first, then with bacteria in mixing cell A, (2) bacteria and 0.4 M NaOH was mixed first, then with 2% SDS in mixing cell A, and (3) bacteria and 2% SDS mixed first, then with 0.4 M NaOH in mixing cell A. For all these tests, the flow rate for all solutions was set to 2.5 cm/s. After the first mixing step, the lysed bacteria were collected, and the remainder of the steps was performed manually. The quantity and quality of the plasmid was examined by agarose gel electrophoresis. The result showed that first mixing NaOH and SDS and then mixing with

Fig. 6 Effect of the shaking frequency. a After the plasmid was extracted from the lysed bacteria with different shaking frequencies, RNA was removed and 5 l of each sample was loaded and run on a 0.7% agarose gel to determine its purity and yield. M The DNA marker, DL15000; C1 plasmid prepared by manual alkaline lysis; C2

plasmid lysed using the device but manually mixed with solution III; lanes 17 lysed bacteria mixed with solution III in the cell B by shaking at frequencies of 0, 35, 45, 55, 65, 75, and 85 Hz. b The intensity of each SC band was analyzed by Quantity One software. Data was representative of three independent experiments

1222 Table 1 Plasmid yield from automated alkaline lysis Plasmids

a

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OD260:280 1.89 1.85 1.82 1.88

Plasmid yield (mg/l) 90.16 80 85.47 83.33

was examined by agarose gel electrophoresis. The results showed that a frequency of 75 Hz produced the highest yield and purity of plasmid DNA (Fig. 6). Determination of the quantity of plasmid DNA To measure the plasmid DNA yield, plasmids pcD-VP1, pEGFP-N3, pcDNA3, and pVAX were prepared by our automated alkaline lysis procedure using the optimized conditions as determined above, and then loaded onto an agarose gel along with a known amount of standard DNA marker. As shown in Table 1, an average of 8090 mg of plasmid DNA was obtained from a 1-l fermentation of bacteria at OD600 nm of 50. Determination of the quality of plasmid DNA

pcDNA3 pVAX1 pcDNA3-VP1 pEGFP-N3

The host E. Coli was DH5 from Invitrogen a Four liters of culture at OD600 nm of 50 was performed for each process. b The plasmid yield was determined after purification.

bacteria produced the highest yield and purity of plasmid DNA (Fig. 3). Effect of mixing cell A To investigate the effect of the presence of mixing cell A and whether it provided a significant improvement in the mixing efficiency of bacteria and solution II, we performed a series of experiments with and without the cell. Bacteria, 0.4 M NaOH, and 2% SDS were pumped at a flow rate of 2.5 cm/s and mixed with or without the presence of cell A. The remaining steps in the processed were performed manually. The quantity and quality of plasmid was examined by agarose gel electrophoresis. The result showed that a higher yield and quality of plasmid DNA was obtained in the group with mixing cell A (Fig. 4). Effects of flow rate The flow rate of the solutions is also a factor affecting bacteria lysis. To find an optimal flow rate, experiments were performed for pumping bacteria, 0.4 M NaOH, and 2% SDS at flow rates of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 cm/s, respectively, for mixing in cell A. The remaining steps in the process were performed manually. The quantity and quality of plasmid was examined by agarose gel electrophoresis. The result showed that a flow rate of 3.0 cm/s produced the highest yield of plasmid DNA (Fig. 5). Effects of shaking frequency The step involving the combination of solution III and the lysed solution from cell A is another important step, as the high viscosity of the lysed solution can impede the mixing of the two solutions. To overcome this problem, the mixing cell B was connected to a horizontal shaker, and the effect of different shaking frequencies was examined. Bacteria, 0.4 M NaOH, 2% SDS, and solution III were all pumped into the apparatus at flow rates of 3.0 cm/s. The experiments were repeated for shaker frequencies of 0, 35, 45, 55, 65, 75 and 85 Hz. The remaining steps in the process were performed manually. The quantity and quality of plasmid

The plasmid quality was analyzed by examining the gel under UV light and measuring its ratio of OD260:280 and by performing a transfection test. As Table 1 shows, the ratio of the OD values of 260 versus 280 nm was 1.82.0, which was within the range of acceptable purity for plasmids. As Fig. 7 shows, the efficiency of transfection was similar to that obtained for samples prepared by the corresponding manual process. Thus, the plasmid prepared in our automated process was of comparable quality to samples obtained manually using previously documented protocols (Sambrook et al. 1989).

Fig. 7 Transfection efficiency. Ten micrograms of pEGFP-N3 extracted by automated alkaline lysis and manual alkaline lysis were transfected into BHK-21 cells. The expression of the green fluorescence was analyzed by FACS. Gray area Negative control, cells without transfection; dashed line transfected with plasmid prepared by the manual alkaline lysis; solid line transfected with plasmid prepared by the automated process. Data was representative of three independent experiments

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Discussion The automated device for alkaline lysis described in this paper provides an automated process for large-scale preparation of plasmid. One of the most important modifications to the process was the elimination of the centrifugation steps, as these represent the most rate-limiting segment for plasmid extraction using the alkaline lysis method. Additionally, we examined the various process parameters to improve the overall efficiency and the quality and yield of the final product. The modular design of the system ensures that the operation can easily be scaled up and experimental parameters can easily be reinvestigated to ensure optimum operation for alternative configurations. To eliminate centrifugations, we employed a 0.2-m hollow fiber cartridge to harvest cells, a filtration system N to remove debris after cell lysis, and a 70-m nylon filter to remove RNA contamination after the plasmid was precipitated in ethanol. Because the hollow fiber cartridge can be manufactured in many different sizes, a suitable cartridge can be selected to recover the desired final volume of fermented bacteria. After mixing with solution III, the lysed bacteria was passed through four layers of nylon filters of gradually decreasing pore size (375, 186, 122, 70 m) to remove debris and other impurities in this size range. The use of a 70-m nylon filter has been observed to be the most effective at separation of RNA from the plasmid after ethanol precipitation. In the plasmid preparation process, the most critical step is probably cell breakage (Lahijani et al. 1996). If the breakage step is too vigorous, the genomic DNA will be broken up and will contaminate the plasmid. If the step is too gentle, less plasmid DNA will be obtained and overall yield will fall. In this protocol, the incorporation of cell A (A in Fig. 1) addresses both of these issues and permits the efficient lysing of the bacteria. By investigating the mixing order of the reagents and the flow rate, we were able to determine the best combination in terms of optimal yield and quality of the final product. An additional problem was that, after mixing with lysing solution II in cell A, the lysed product was very viscous and difficult to mix with solution III. This mixing step is also an important part in the process, as the alkalinity of the lysed product must be reduced rapidly to avoid secondary reactions that can adversely affect the quality of the final product. We overcame this problem by including a second mixing cell B, combined with a shaker, to assist in the mixing process. By examining a range of frequencies for the shaker, we were able to determine which produced the best results for the final product in terms of quality and yield. The procedure provides a fast and efficient way to extract plasmid in large volumes. After extracting plasmid with the described protocol and automated device, the plasmid may be purified using any available purification

protocol. We have compared the extracted product obtained from our system to the product obtained from a manual alkaline lysis, both purified in the Qiagen plasmid purification columns (Qiagen, Germany), and found comparable transfection efficiency in both samples.

Acknowledgment This work was supported in part by the China Key Technologies R&D programme (2004AA213102 and 2003AA241110) and a research initiative fund provided to B.W. by CAU. We would also like to thank Dr. QL Yu for her assistance with the work.

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