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.     

To Plate or to Simply Unfreeze,That Is the Question for Optimal Plasmid Extraction

Many molecular biology applications require fast plasmid DNA extraction, spurring multiple studies on how to speed up the process. It is regularly instructed in standard laboratory protocols to plate out frozen glycerol bacterial stocks prior to bacteria incubation in liquid media and subsequent plasmid extraction, although the rationale for this is often unexplained (other than for the isolation of single colonies). Given the commonality and importance of this laboratory operation, such a practice is time-consuming and laborious. To study the impact of this practice and the alternative direct culturing method, we investigated the association between bacterial cell mass and its potential influence on plasmid yields from the 2 methods. Our results showed no difference with preplating for 7 out of 8 plasmid constructs used in the study, suggesting that direct glycerol recovery would not lead to poorer plasmid yields. The findings support the rationale for direct glycerol recovery for plasmid extraction, without the need of an intermediate preplating step.     

Electrooptics studies of Escherichia coli electropulsation: orientation, permeabilization, and gene transfer.

Fast optical transient signals are suitable approaches to the investigation of the behavior of bacteria during an electric pulse. In a previous work, by a dual approach taking advantage of a video method and a fast kinetic study of the light transmitted across a cell suspension, we showed that a field-induced orientation phenomenon was affecting the rod-shaped bacteria during the pulse (Eynard et al., 1992. Eur. J. Biochem. 209:431-436). In the present work, time courses of electro-induced responses of bacteria during a single square-wave pulse are analyzed. Observations of both the orientation step and the permeabilization process are relevant. These two steps are affected by the addition of DNA. They both obey to a first-order kinetic. The conclusion of this work is that Escherichia coli permeabilization and transformation are multistep processes: orientation (step 1) is followed by an envelope alteration (step 2), all steps being affected by plasmid addition. In the case of E. coli, a rod-shaped bacteria, the orientation process (step 1) brings the cell parallel to the field direction. The pulse duration must be longer than the orientation characteristic time (approximately 1 ms) to trigger an effective permeabilization and its associated events. The permeabilization process (step 2) is associated with a field-induced dipole effect.     

Enolase is present in the cell wall of Saccharomyces cerevisiae

We have reported that the macrophage-like cell line J774.1, when infected with the periodontopathic bacterium Actinobacillus actinomycetemcomitans, undergoes apoptosis. In this study, we examined whether stimulation of J774.1 cells with lipopolysaccharide (LPS) before the infection affects the subsequent apoptosis. Cytotoxicity on the LPS-stimulated cells was about half of the unstimulated control cells. DNA fragmentation in the LPS-stimulated cells was also significantly lower than in the control cells, whereas it was increased to a level similar to that of the control cells by addition of a nitric oxide (NO) inhibitor. In addition, significantly smaller numbers of live A. actinomycetemcomitans were recovered from the LPS-stimulated macrophages at 8 h after the infection as compared with the control cells. These findings suggest that the inhibitory effect of LPS on apoptosis results from an enhanced NO-mediated bactericidal activity.     

一种简便易行核酸疫苗质粒纯化工艺的开发

介绍了一种成本低、步骤少、简单易行的质粒纯化制检工艺。该工艺选择优势产生超螺旋质粒的大肠杆菌菌株以无蛋白质培养基进行发酵罐培养,采用碱裂解法,对质粒制备过程中所用的层析吸附材料、核酸结合溶液、去除内毒素等杂质的方法和浓缩等步骤进行了实用性改进,并建立了相应的检定方法,所得质粒的纯度达到临床级要求。     

Plasmid DNA binds to the core oligosaccharide domain of LPS molecules of E. coli cell surface in the CaCl2-mediated transformation process

In the standard procedure for artificial transformation of E. coli by plasmid DNA, cellular competence for DNA uptake is developed by suspending the cells in ice-cold CaCl2 (50-100 mM). It is believed that CaCl2 helps DNA adsorption to the lipopolysaccharide (LPS) molecules on E. coli cell surface; however, the binding mechanism is mostly obscure. In this report, we present our findings of an in-depth study on in vitro interaction between plasmid DNA and E. coli LPS, using different techniques like absorption and circular dichroism spectroscopy, isothermal titration calorimetry, electron and atomic force microscopy, and so on. The results suggest that the Ca(II) ions, forming coordination complexes with the phosphates of DNA and LPS, facilitate the binding between them. The binding interaction appears to be cooperative, reversible, exothermic, and enthalpy-driven in nature. Binding of LPS causes a partial transition of DNA from B- to A-form. Finer study with the hydrolyzed products of LPS shows that only the core oligosaccharide domain of LPS is responsible for the interaction with DNA. Moreover, the biological significance of this interaction becomes evident from the observation that E. coli cells, from which the LPS have been leached out considerably, show higher efficiency of transformation, when transformed with plasmid-LPS complex rather than plasmid DNA alone.     

Recombination-dependent concatemeric plasmid replication.

The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector. Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination. Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described. Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication. On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells. The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes.     

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.     

Molecular cloning of a gene encoding a Chlamydia psittaci 57-kDa protein that shares antigenic determinants with ca. 60-kDa proteins present in many Gram-negative bacteria

In order to develop reagents to study the immune response of guinea pigs to infection by Chlamydia psittaci guinea pig inclusion conjunctivitis strain (GPIC), we constructed a plasmid clone bank with C. psittaci DNA. One of the recombinant clones isolated produced large amounts of a 57-kilodalton (kDa) protein that was immunoreactive with sera from GPIC infected guinea pigs. While investigating this recombinant protein, we discovered that all the Gram-negative bacteria analyzed so far have immunoreactive proteins of similar size. This protein seems to be a 'common antigen' already described in various Gram-negative bacteria.     

Synthesis of lipopolysaccharides in the bacterium Yersinia pseudotuberculosis: effect of the pVM82 plasmid and growth temperature

The composition and structure of lipopolysaccharides (LPS) of three isogenic strains of Yersinia pseudotuberculosis serovar O:1b (without plasmids (82-) and with plasmids pVM82 (82+) or p57 (57+)) grown at 8 or 37 degrees C were studied by chemical and immunochemical methods, SDS-polyacrylamide gel electrophoresis, and 13C-NMR spectroscopy. At the lower temperature, the (82-) and (82+) strains synthesized S-form of LPS with similar structure characterized by high acylation and immunochemical activity. On the other hand, LPS of the (82+) strain had shorter carbohydrate chains than LPS of the (82-) strain. The contents of LPS were decreased in cells of the plasmid-free strain grown at the higher temperature. LPS isolated from these cells were of the R-form and had low acylation and immunochemical activity. Total LPS content in cells of the (82+) strain did not significantly depend on the growth temperature. LPS of the warm variant of these bacteria contained a polysaccharide fragment and had moderate immunochemical activity. The cells of the (57+) strain at both growth temperatures had low LPS contents and produced LPS of low acylation without O-specific chains (cold variant) or containing O-polysaccharide with low polymerization degree (bacteria grown at 37 degrees C). The data indicate that in the absence of the plasmids, LPS synthesis is encoded by the chromosomal genes in pseudotuberculosis bacteria. Expression of the genes involved in LPS synthesis is regulated by the temperature of bacterial growth. Genes responsible for temperature-dependent regulation of LPS biosynthesis are located on chromosomal DNA. The pVM82 plasmid includes two gene groups; one group is localized in a 57-mD fragment of DNA and inhibits LPS synthesis, suppressing temperature-dependent regulation of the synthesis. The genes located in a 25-mD fragment of the pVM82 plasmid are de-repressors of the 57-mD fragment, and they restore the ability of pseudotuberculosis bacteria to synthesize relatively long LPS at both growth temperatures.     

A continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF

Plasmid DNA for biopharmaceutical applications is produced easily in Escherichia coli bacteria. The cell lysis is the most crucial step for purification of plasmid DNA. In this paper, we describe a continuous cell alkaline lysis, neutralization, and clarification combination process for production of plasmid pUDK-HGF using hollow fiber ultrafiltration column as a lysis chamber and compare the plasmid DNA yield and homogeneity with the T-connector and manual processes, respectively. The results show that the plasmid pUDK-HGF yield of the combination process is 13% higher than manual lysis, twice higher than using T-connector. When the proportion of lysed cells and neutralization solution is 3:1, the plasmid pUDK-HGF yield can improve by 70%. This process could be easily scaled up to meet the industrial scale for cell lysis.     

Adsorptive purification of pDNA on superporous rigid cross-linked cellulose matrix

Use of plasmid DNA (pDNA) in the emerging gene therapy requires pure DNA in large quantities requiring production of safe DNA on large scale. While a number of kit-based DNA purification techniques have become popular, large scale cost effective purification of DNA remains a technological challenge. Most traditional, as well as newly developed methods for DNA purification are expensive, tedious, use toxic reagents, and/or generally not amenable for scaled up production. Our attempts to develop a scalable adsorptive separation technology resulted in successful use of indigenously developed rigid cross-linked cellulose beads for single step purification of pDNA from alkaline cell lysates. This mode of purification employs a combination of intra-particle interactions that could give a product plasmid DNA free from chromosomal DNA, RNA and host proteins in a single scalable chromatographic step. The technology can be employed as a batch adsorption step on small scale, or on a large scale column chromatography. A high copy number 9.8 kb plasmid (from an Escherichia coli strain) was purified in yields of 77 and 52%, respectively in batch and column modes. The product obtained was homogeneous supercoiled plasmid with no RNA and protein contamination confirmed by quantitative analysis, agarose gel electrophoresis and SDS-PAGE.     

Contaminants within bacterial plasmid preparations trigger apoptosis in liposome transfected OVCAR3, but not in SKOV3 or AZ224 human ovarian cancer cells

Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.     

Cutting edge: bacterial DNA and LPS act in synergy in inducing nitric oxide production in RAW 264.7 macrophages.

LPS is well recognized for its potent capacity to activate mouse macrophages to produce NO, an important inflammatory mediator in innate host defense. We demonstrate here that, although inducing little NO alone, DNA from both Gram-negative and Gram-positive bacteria synergizes with subthreshold concentrations of LPS (0.3 ng/ml) to induce NO in cultures of RAW 264.7 macrophages. The effects of the DNA are mimicked by synthetic CpG-containing oligodeoxynucleotides but not by non-CpG-containing oligodeoxynucleotides. This synergistic activity is not inhibited by neutralizing Abs against IFN. Preincubation of macrophages with DNA for 8-24 h suppresses subsequent synergistic macrophage responses to DNA/LPS, whereas prolonged pretreatment with LPS enhances synergy. RT-PCR analysis indicates that the mRNA levels of the inducible NO synthase gene are also coordinately suppressed or induced. These findings indicate that temporally controlled, synergistic interactions exist between microbial DNA and LPS in the induction of macrophage NO via enhanced inducible NO synthase gene expression.     

The opposing effects of lipopolysaccharide on the antitumor therapeutic efficacy of DNA vaccine

DNA vaccine represents a novel method to elicit immunity against infectious disease. Lipopolysaccharide (LPS) copurified with plasmid DNA may affect therapeutic efficacy and immunological response. We aimed to study the effect of LPS on the therapeutic efficacy of HER-2/neu DNA vaccine in a mouse tumor animal model. Plasmid DNA purified from commercial EndoFree plasmid purification kits functioned as a better therapeutic DNA vaccine than that purified from Non-EndoFree purification kit, which contains >or=0.5 microg LPS per 100 mg DNA plasmid. To further investigate the effect of LPS on the therapeutic efficacy of DNA vaccine, increasing amount of LPS was added to endotoxin-free plasmid DNA, and inoculated on mice with established tumors. One mug of LPS significantly attenuated the therapeutic effect of neu DNA vaccine and increased Th2 immune responses bias with interleukin-4 cytokine production. In contrast, high amount (100 microg) of LPS enhanced the therapeutic efficacy of neu DNA vaccine with an increase of cytotoxic T lymphocyte response and Th1 immune response. The effect of LPS on DNA vaccine was diminished when the tumor was grown in toll-like receptor 4 (TLR4)-mutant C3H/HeJ mice. Our results indicate that variation in the LPS doses exerts opposing effects on the therapeutic efficacy of DNA vaccine, and the observed effect is TLR4 dependent.     

An extended boiling method for small-scale preparation of plasmid DNA tailored to long-range automated sequencing

Automated fluorescence sequencing depends on high-quality plasmid DNA, which is conveniently prepared by minipreparation procedures. While those procedures are effective for high-copy number plasmids, purity and yields of low-copy number plasmids are often not sufficient to achieve reasonable sequencing results. Here, we describe a reproducible and cheap procedure for the small-scale preparation of plasmid DNA, which is based on the original Holmes and Quigley protocol, comprising a boiling and two selective precipitation steps. Besides various other modifications, this procedure utilizes polyethylene glycol (PEG) precipitation as a key step to further purify plasmid DNA tailored to automated fluorescence sequencing. Independent of the plasmid size and copy number, the modified procedure yields plasmid DNA, which gives average reading lengths of 800 and more bases with a standard fluorescence cycle sequencing protocol. To demonstrate the efficiency and reproducibility of the method, sequencing data of various human interleukin-6 gene variants cloned in different vectors are presented. This procedure offers an economical alternative to commercial miniprep kits, utilizing silica resins or anion-exchanger matrices and, moreover, is more reliable and consistent with respect to reading lengths and accuracy in automated fluorescence sequencing.     

Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

The use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter, we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers, which exhibit deleterious leakage under similar conditions. Furthermore, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2 M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.     

A method for the isolation of covalent DNA-protein crosslinks suitable for proteomics analysis

The covalent crosslinking of protein to DNA is a form of DNA damage induced by a number of commonly encountered agents, including metals, aldehydes, and radiation as well as chemotherapeutic drugs. DNA-protein crosslinks (DPCs) are potentially bulky and helix distorting and have the potential to block the progression of translocating protein complexes. To fully understand the induction and repair of these lesions, it will be important to identify the crosslinked proteins involved. To take advantage of dramatic improvements in instrument sensitivity that have facilitated the identification of proteins by proteomic approaches, improved methods are required for isolation of DPCs. This article describes a novel method for the isolation of DPCs from mammalian cells that uses chaotropic agents to isolate genomic DNA and stringently remove noncrosslinked proteins followed by DNase I digestion to release covalently crosslinked proteins. This method generates high-quality protein samples in sufficient quantities for analysis by mass spectrometry. In addition, the article presents a modified form of this method that also makes use of chaotropic agents for promoting the adsorption of DNA (with crosslinked proteins) to silica fines, markedly reducing the DPC isolation time and cost. These approaches were applied to radiation- and camptothecin-induced DPCs.     

Rapid isolation of high quality, multimeric plasmid DNA using zwitterionic detergent

Purification of plasmid DNA from bacteria is an essential tool in recombinant DNA technology and has become an essential task in laboratories to industries. Moreover, the recent progress of "Gene therapy" and "Genetic vaccination" also demands production of pharmaceutical grade plasmid DNA in 'kilogram' level. Despite existence of a number of purification protocols, all most all have been originated from a pioneering work [Birnboim, H.C., Doly, J., 1979. A rapid extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 1513-1523] and so suffer from one or more drawbacks, such as purification time, purity or quantity of isolated plasmid DNA. Here, we have reported an innovative approach for isolation of highly pure and functional plasmid DNA in significant amount, based on generation of "soft protein aggregate" with the help of zwitterionic detergents and alkali. Solibilized proteins and RNA could be removed by a simple and mild washing with Tris buffer of low ionic strength and multimeric plasmid DNA could be eluted in a single step from the protein aggregate. Additionally, isolated plasmid DNA could easily be digested by restriction enzymes and had high functionality in protein expression. Thus, considering both its remarkable simplicity and efficiency in producing sufficiently pure plasmid DNA, the new strategy would emerge a useful tool in modern recombinant technology and therapeutic applications.