Coupled chromatography on Sepharose 4B-cellulose nitrate columns for the isolation of an R-plasmid fromPseudomonas aeruginosa

A chromatographic technique for isolating bacterial plasmid DNA using a Sepharose 4B column connected to a cellulose nitrate column is described. After loading the Sepharose 4B column with the alkali-treated cleared bacterial lysate, the plasmid DNA was eluted from the cellulose nitrate column by passing 2 M NaCl-0.01 M sodium citrate buffer through the connected columns. This one-step procedure allows separation of plasmid DNA at a concentration suitable for direct electron microscopy. This method was applied to the separation and electron microscopic examination of a newPseudomonas aeruginosa plasmid (pYMB1).     

Precipitation by polycation as capture step in purification of plasmid DNA from a clarified lysate

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Large-scale purification of plasmid DNA from bacterial cell culture normally includes one or several chromatographic steps. Prechromatographic steps include precipitation with solvents, salts, and polymers combined with enzymatic degradation of nucleic acids. No method alone has so far been able to selectively capture plasmid DNA directly from a clarified alkaline lysate. We present a method for selective precipitation of plasmid DNA from a clarified alkaline lysate using polycation poly(N, N'-dimethyldiallylammonium) chloride (PDMDAAC). The specific interaction between the polycation and the plasmid DNA resulted in the formation of a stoichiometric insoluble complex. Efficient removal of contaminants such as RNA, by far the major contaminant in a clarified lysate, and proteins as well as 20-fold plasmid concentration has been obtained with about 80% recovery. The method utilizes a inexpensive, commercially available polymer and thus provides a capture step suitable for large-scale production.     

Isolation of plasmid DNA from cell lysates by aqueous two-phase systems

This work presents a study of the partitioning of a plasmid vector containing the cystic fibrosis gene in polyethylene glycol (PEG)/salt (K2HPO4) aqueous two-phase systems (ATPS). The plasmid was extracted from neutralized alkaline lysates using PEG with molecular weights varying from 200 to 8000. The effects of the lysate mass loaded to the ATPS (20, 40, and 60% w/w) and of the plasmid concentration in the lysate were evaluated. The performance of the process was determined by qualitative and quantitative assays, carefully established to overcome the strong interference of impurities (protein, genomic DNA, RNA), salt, and PEG. Plasmid DNA partitioned to the top phase when PEG molecular weight was lower than 400. The bottom phase was preferred when higher PEG molecular weights were used. Aqueous two-phase systems with PEG 300, 600, and 1000 were chosen for further studies on the basis of plasmid and RNA agarose gel analysis and protein quantitation. The recovery yields were found to be proportional to the plasmid concentration in the lysate. The best yields (>67%) were obtained with PEG 1000. These systems (with 40 and 60% w/w of lysate load) were able to separate the plasmid from proteins and genomic DNA, but copartitioning of RNA with the plasmid was observed. Aqueous two-phase systems with PEG 300 concentrated both plasmid and proteins in the top phase. The best system for plasmid purification used PEG 600 with a 40% (w/w) lysate load. In this system, RNA was found mostly in the interphase, proteins were not detected in the plasmid bottom phase and genomic DNA was reduced 7.5-fold.     

Purification of plasmid DNA using tangential flow filtration and tandem anion-exchange membrane chromatography

A new bioprocess using mainly membrane operations to obtain purified plasmid DNA from Escherechia coli ferments was developed. The intermediate recovery and purification of the plasmid DNA in cell lysate was conducted using hollow-fiber tangential filtration and tandem anion-exchange membrane chromatography. The purity of the solutions of plasmid DNA obtained during each process stage was investigated. The results show that more than 97% of RNA in the lysate was removed during the process operations and that the plasmid DNA solution purity increased 28-fold. One of the main characteristics of the developed process is to avoid the use of large quantities of precipitating agents such as salts or alcohols. A better understanding of membrane-based technology for the purification of plasmid DNA from clarified E. coli lysate was developed in this research. The convenience of anion-exchange membranes, configured in ready-to-use devices can further simplify large-scale plasmid purification strategies.     

Enhanced purification of plasmid DNA using Q-Sepharose by modulation of alcohol concentrations

Ion-exchange chromatography is one of the most commonly used methods for plasmid preparation. In this study a modified method was used to purify plasmid from bacterial lysate using Q-Sepharose. Incorporation of alcohols into the washing buffers enhanced the separation of plasmid from RNA and proteins. The use of isopropanol and ethanol achieved a high yield and purity whereas the use of methanol failed to improve the plasmid purification using Q-Sepharose by batch adsorption-desorption. Stepwise elution containing various concentrations of isopropanol and NaCl was used in preparative chromatography to enhance the plasmid purification. The same stepwise elution was applied to the chromatography columns packed with 0.5, 20, and 200 ml of Q-Sepharose for plasmid purification from 7.5, 300, and 3000 ml bacterial broth, respectively. Complete separation of DNA from RNA and proteins was achieved under gravity flow by modulation of the alcohol concentrations in the stepwise elution. These three scales of chromatography maintained an approximate plasmid yield and the purified plasmid contained undetectable levels of RNA and protein.     

Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clarified alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid purification. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clarified alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 7g/ml to 20 7g/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.     

体积排阻液相色谱法质粒的含量测定

首次以体积排阻液相色谱法进行质粒提取液浓度的测定研究,通过纯质粒溶液在260 nm处的紫外吸峰面积与质粒浓度之间数据的线性最小二乘拟合,得到了反映质粒浓度和紫外吸收峰峰面积之间关系的标准曲线:Y=2×10~(-5) X,通过验证实验,确定了质粒提取液中不同组分,如核糖核酸,蛋白质等在色谱图中的出峰位置.结合标准曲线测定了自制的质粒提取液的质粒浓度为0.32 mg/mL.该方法方便,快捷,准确.     

Polyelectrolyte complexes as a tool for purification of plasmid DNA. Background and development

The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration.     

Separation of supercoiled from open circular forms of plasmid DNA,and biological activity detection

To establish a cost-effective purification process for the large-scale production of plasmid DNA for gene therapy and DNA vaccination, a single anion-exchange chromatography (AEC) step was employed to purify supercoiled plasmid DNA (sc pDNA) from other isoforms and Escherichia coli impurities present in a clarified lysate. Two different size and conformation plasmids were used as model targets, and showed similar elution behavior in this chromatographic operation, in which sc pDNA was effectively separated from open circle plasmid DNA (oc pDNA) in a salt gradient. The process delivered high-purity pDNA of homogeneity of 95 ± 1.1% and almost undetectable levels of endotoxins, genomic DNA, RNA and protein, at a yield of 65 ± 8%. Furthermore, the transfection efficiency (29 ± 0.4%) was significantly higher than that (20 ± 0.1%) of a pDNA control. The present study confirms the possibility of using a single AEC step to purify sc pDNA from other isoforms and host contaminants present in a clarified E. coli lysate.     

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.     

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.     

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.     

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated lipopolysaccharides (LPS) contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high-quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive, and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures.     

Contaminant eluted from solid-phase plasmid affinity-purification protocol columns is not found using liquid-phase methods and can be prevented.

The preparation of high quality plasmid DNA is a necessary requirement for most molecular biology applications. We compared four different large plasmid preparation protocols, which were based on either a liquid-phase approach (Triton lysis) or purification of alkaline lysis bacterial extracts followed by supercoiled plasmid purification on affinity columns. Two host Escherichia coli strains, JM 109 and INValphaF', were used to grow the test plasmids for comparison of product plasmid DNA produced from the four different plasmid isolation methods. While the DNA grown in E. coli strain JM109, prepared by liquid-phase Triton lysis was appropriately restricted by 12 restriction enzymes, this was not the case for any of the JM109-grown DNA purified by any of the affinity column solid-phase approaches. In contrast to this, when the plasmid DNA was grown in E. coli strain INValphaF', most restriction enzymes cut DNA appropriately, irregardless of the plasmid preparation protocol used. It seems that an impurity commonly eluted with the DNA from all three of the solid-phase DNA columns had an equal effect on the above enzymes using the common host strain JM109, but not strain INValphaF'.     

A new method of plasmid DNA preparation by sucrose-mediated detergent lysis from Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive)

A simple and cheap method of plasmid DNA preparation from both gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) organism is presented here. In this method, in place of the high-priced chemicals lysostaphin and lysozyme which are commonly used for removal of cell-wall during plasmid DNA preparation from gram-positive and gram-negative bacteria, respectively, only sucrose has been used. Firstly, bacteria is treated with Trizma (pH 8.0) containing 100% sucrose (hypertonic solution). Due to this osmotic shock, protoplasm covered by the plasma membrane of bacteria possibly shrinks and becomes detached from the cell-wall. Osmotically sensitive cells thus formed, from gram-positive (S. aureus) and gram-negative (E. coli) bacteria, are finally lysed by the lysis mixture, containing brij 58 and sodium deoxycholate. The lysate is centrifuged at 15,000 rpm for 30 min to pellet the cell debris. The supernatant containing plasmid DNA is treated with either polyethylene glycol or isopropanol. The precipitate which contains plasmid DNA is dissolved in a buffer containing Tris, EDTA, NaCl, and sodium dodecyl sulfate (pH 8.0); thus protein is denatured and removed. Finally, RNA is removed by RNase treatment. The average yield of staphylococcal plasmid DNA as well as plasmid pBR322 from E. coli HB101 in 100% sucrose-treated preparations is greater than that of lysostaphin- and lysozyme-treated preparations. This method is applicable for both large-scale and small-scale preparations. The substrate activity for restriction enzyme, cloning, transforming ability, and electron microscopic profile of the plasmid DNA prepared by this method remains unaltered.     

Reverse micellar extraction systems for the purification of pharmaceutical grade plasmid DNA

Plasmid DNA as an active pharmaceutical ingredient (API) is gaining more and more importance. For the production of multigram quantities of this substance robust and scalable processes comprising several purification steps have to be designed. One main challenge is the initial separation of plasmid DNA and RNA in such a purification scheme. In this study we investigated the distribution of plasmid DNA and RNA in reverse micellar two-phase systems which is considered to be the basis for the development of an extractive purification step that can easily be integrated into common processes. For this purpose the distribution of the 4.6kb plasmid pUT649 and Escherichia coli RNA in systems comprising isooctane, ethylhexanol, and the surfactant methyltrioctylammoniumchloride (TOMAC) under the influence of different salts was studied. Anion concentrations at which the partitioning behaviour for nucleic acids inverted (inversion point) were identified. Systems capable of separating RNA from plasmid DNA were further analysed and applied to extract RNA from plasmid DNA out of a preconditioned cleared lysate. The capability of reverse micellar systems for plasmid form separation was also shown by capillary and agarose gel electrophoresis.     

Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection

DNA extracted and purified for vaccination, gene therapy or transfection of cultured cells has to meet different criteria. We describe herein, a scalable process for the primary extraction of plasmid DNA suitable for transient expression of recombinant protein. We focus on the scale up of alkaline lysis for the extraction of plasmid DNA from Escherichia coli, and use a simple stirred tank reactor system to achieve this. By adding a series of three precipitations (including a selective precipitation step with ammonium acetate) we enrich very quickly the plasmid DNA content in the extract. The process has been thus far used to extract up to 100 mg of plasmid from 1.5 l of clarified lysate, corresponding to an E.coli bioreactor fermentation of 3 l. This revised version was published online in July 2006 with corrections to the Cover Date.     

A continuous method for the large-scale extraction of plasmid DNA by modified boiling lysis

This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, a modification of the basic boiling lysis technique, to simplify the processing of large volumes of Escherichia coli cultures. Fermented bacteria are harvested using a hollow fiber-membrane module and pre-treated with lysozyme prior to passing through a thermal exchange coil set at 70 degrees C to lyse the cells, and into a juxtaposed cooling coil on ice. The lysed and cooled bacteria are subsequently separated from the lysate by centrifugation and plasmid DNA is precipitated from the supernatant for further purification. The use of peristaltic pumps and two heating coils at constant temperature without the use of centrifugation enable the lysis process to become constant and controllable, providing a flow-through protocol for cell lysis and plasmid DNA extraction. Large volumes of bacterial cultures (20 l) can be processed in 2 h, yielding approximately 100 mg plasmid DNA l(-1) culture, making this an attractive protocol for consistent and large-scale preparation of plasmid DNA.