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.     

Rapid purification of plasmid DNAs by hydroxyapatite chromatography.

A method is described for the rapid preparation of plasmid DNAs of molecular weight up to 14 X 10(6). This method involves the chromatography, at room temperature, of bacterial cleared lysates on hydroxyapatite in the presence of high concentrations of phosphate and urea. All detectable protein and RNA contamination of plasmid DNA is removed by this procedure and the conformation of the plasmid DNA is unaffected. Less than 0.5% chromosomal DNA is present in the purified preparation and even this can be removed if necessary by a simple extention of the procedure to include a heat-denaturation step. The method is extremely rapid and amenable to large-scale plasmid preparation; 5 mg ColE1 DNA have been purified within 40 min. The yield of plasmid DNA is similar to that obtained with the conventional dye-centrifugation technique, however the purity is greater.     

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.     

Preparative purification of plasmid DNA templates for in vitro transcription assays by consecutive differential precipitations

A procedure for large-scale isolation of plasmid DNA without the use of RNase has been developed to obtain a DNA template for preparative in vitro RNA synthesis catalyzed by phage RNA polymerases. The separation of plasmid DNA from admixtures has been achieved only through selective precipitations of either plasmid DNA or contaminants. No expensive reagents or equipment were required. Plasmid quality was evaluated by gel electrophoresis and restriction analysis. The obtained plasmid DNA templates have been shown to be devoid of any detectable ribonucleolytic activity that may interfere with the following RNA synthesis.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A rapid and inexpensive method for preparing E. coli plasmid-DNA

A simple, rapid and inexpensive scaled up miniprep procedure for preparing pure E. coli plasmid DNA is described. Bacterial cells were subjected to the boiling procedure and high molecular weight RNA was removed by LiCl-precipitation. Residual RNA and proteins were removed by subsequent treatment with RNase A and proteinase K/SDS respectively, followed by Sephadex G-50 and Sepharose 6B-Cl chromatography. The average yield from a 100 ml over-night bacterial suspension was 100 to 150 micrograms for pBR-322 DNA, and 250-500 micrograms for SP-6 derived recombinant plasmids. In addition, the described "scaled up" preparation does not require CsCl-ethidium bromide centrifugation; pure plasmid DNA can be prepared within 1 to 2 days.     

A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder

A preparative procedure for obtaining highly purified plasmid DNA from bacterial cells is described. The method is adapted from our earlier procedure, which gave partially purified plasmid in a form suitable for rapid screening of a large number of samples. In the present method, all detectable RNA, chromosomal DNA, and protein are removed without the use of enzymes, phenol extraction, dialysis, or equilibrium centrifugation. Binding of plasmid DNA to glass powder in the presence of 6 m sodium perchlorate is used for the final purification step.     

Generation of chromosomal DNA during alkaline lysis and removal by reverse micellar extraction

The separation of structurally related impurities from pharmaceutical plasmid DNA by highly scalable purification techniques is a challenge for biochemical engineering. Next to RNA, proteins, and lipopolysaccharides, the chromosomal DNA of the plasmid replicating host has to be removed. Here, we describe the application of reverse micellar extraction for the separation of chromosomal from plasmid DNA. By applying different procedures for alkaline lysis, bacterial lysates with different amounts of chromosomal DNA were generated. A reverse micellar extraction step enabled us to deplete the concentration of this impurity below the required level of 50 mg g⁻¹ of plasmid DNA with almost complete plasmid recovery.     

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.     

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.     

A rapid miniprep for the isolation of total DNA fromAgrobacterium tumefaciens

A procedure which avoids the use of phenol-chloroform and RNAase for the isolation of total DNA fromA. tumefaciens is described. Specific precipitation of protein by 2.5 M ammonium acetate is employed and much of the RNA is removed by an isopropanol precipition step. The procedure yields easily restrictable, good quality DNA and is probably applicable to other Gramnegative bacteria.     

Purification of RNA-free plasmid DNA using alkaline extraction followed by Ultrogel A2 column chromatography

A procedure for extracting RNA-free plasmid DNA from bacterial cells is described. The method is simple and rapid enough to obtain pure plasmid DNA in 8 to 10 h after plasmid amplification. The protocol uses the alkaline extraction procedure described by Birnboim and Doly (1979, Nucl. Acid Res. 7, 1513-1523). Plasmid DNA is then separated from high-molecular-weight RNA by ammonium acetate precipitation and from low-molecular-weight RNA contaminants by Ultrogel A2 column chromatography. The plasmid DNA obtained by this inexpensive technique is sufficiently pure to be used for restriction endonuclease analysis, 5'-end labeling, S1 mapping, DNA sequencing, and colony hydridization.     

Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.     

Rapid and simple removal of contaminating RNA from plasmid DNA without the use of RNase

A procedure for the removal of RNA and RNA fragments from large quantities of pBR322 plasmid DNA without the use of RNase is described. Sephacryl S-300 is employed for the separation of low-molecular-weight RNA from plasmid DNA molecules on the basis of gel filtration. The technique thus circumvents many of the dangers associated with treating plasmid DNA preparations with RNase. The procedure should be generally applicable to the purification of virtually any type of plasmid DNA isolated from a bacterial host.     

Affinity purification of plasmid DNA directly from crude bacterial cell lysates

We have shown previously that a sequence-specific DNA-binding protein based on the Lac repressor protein can isolate pre-purified DNA efficiently from simple buffer solution but our attempts to purify plasmids directly from crude starting materials were disappointing with impractically low DNA yields. We have optimized the procedure and present a simple affinity methodology whereby plasmid DNA is purified directly by mixing two crude cell lysates, one cell lysate containing the plasmid and the other the protein affinity ligand, without the need for treatment by RNaseA. After IMAC chromatography, high purity supercoiled DNA is recovered in good yields of 100-150 microg plasmid per 200 mL shake flask culture. Moreover, the resulting DNA is free from linear or open-circular plasmid DNA, genomic DNA, RNA, and protein, to the limits of our detection. Furthermore, we show that lyophilized affinity ligand can be stored at room temperature and re-hydrated for use when required.     

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.     

A simple procedure for large-scale purification of plasmid DNA

We report a simple, rapid and reliable procedure for large-scale purification of plasmid DNA from non-amplified bacterial cultures. It is a modification of the boiling method of Holmes and Quigley [Anal. Biochem. 114 (1981) 193-197] and involves gel-filtration chromatography using Sephacryl S-1000 for final purification of plasmid DNA. This method does not require CsCl gradients and the recovered plasmids are free of RNA and chromosomal DNA, are supercoiled, retain their biological activity, and are suitable for restriction analysis.     

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.     

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.     

基因治疗用质粒DNA生产面临的问题及研究进展

基因治疗已成为21世纪一些重大疾病的有效治疗策略,目前携带治疗基因的重组质粒已作为基因药物进入临床研究。对用于基因治疗的生物制品的生产与质量控制都有相当严格的要求。虽然已建立大规模符合药学规格的质粒DNA生产工艺,能满足临床需求,但在这些生产工艺中还存在一些难以克服的瓶颈,如:载体构建、细胞裂解、细菌染色体DNA去除、细菌内毒素去除、生产过程中质量控制等。就近年来大规模生产临床用质粒DNA遇到的相关问题及解决方案作一综述。