Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.     

Binding and purification of plasmid DNA using multi-layered carbon nanotubes

We propose a new method for the separation of nucleic acids using multi-layered carbon nanotubes (CNTs) as an adsorbent. According to agarose gel electrophoresis, oxidized water-stable CNTs adsorb certain forms of nucleic acids, such as high molecular weight RNA, chromosomal DNA, linear and denatured forms of plasmid DNA. However, CNTs do not adsorb supercoiled form of plasmid DNA. Nucleic acids bound to CNTs can be readily removed by centrifugation whereas supercoiled plasmid DNA remains in solution. Upon the addition of divalent metal ions supercoiled plasmid DNA forms relatively stable complexes with CNTs due to chelation. Thus, new details about association of nucleic acids with CNTs were revealed and stoichiometry of the complexes was estimated. Our results can be used for fine purification of supercoiled plasmid DNA for gene therapy applications as well as manipulation of nucleic acids for biosensor design.     

Specific recognition of supercoiled plasmid DNA in arginine affinity chromatography

Arginine chromatography was used to fully separate supercoiled and open circular plasmid DNA (pDNA) isoforms. The results show that the arginine matrix promotes multiple interactions with pDNA, including not only electrostatic and hydrophobic but also biorecognition of nucleotide bases by the arginine ligand. The strong interactions occurring with DNA backbone provide stability, conducting to high effectiveness of arginine support to bind pDNA at low ionic strength. The specific interaction of arginine with sc pDNA could be due to the ability of arginine matrix to be involved in complex interactions that are partly dependent on the conformation of the DNA molecule.     

Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption

Separation of the different plasmid isoforms is a major challenge in purifying plasmid DNA. We describe a new type of biochemical interaction that occurs in the presence of high concentrations of lyotropic salt and results in the selective adsorption of supercoiled plasmid DNA to aromatic thioether ligands. Under well-defined conditions, these ligands are capable of separating supercoiled plasmid DNA (ccc) from its isoform, i.e. open circular (oc) form. Integrated in a process, preceded by group separation and followed by anion-exchange chromatography, this new purification method may facilitate the production of highly purified supercoiled plasmid DNA for use in gene therapy and DNA vaccine applications.     

Plasmid adsorption to anion-exchange matrices: comments on plasmid recovery

A number of anion-exchange adsorbents were constructed, employing nonporous silica fibers, and examined with the aim of describing factors that influence desorption and recovery of plasmid DNA (pDNA). The fibers were provided with ligands via adsorption of the polymeric amines poly(ethyleneimine) or chitosan, or via graft-polymerization of primary, tertiary, or quaternary amine monomers to vinyl-silanized fibers. Several adsorbents showed an almost irreversible plasmid binding. It was suggested that important factors affecting the DNA releasing ability are (i) type of amine ligand used (primary amines bind plasmids the strongest), (ii) the structure of the nucleic acid (supercoiled pDNA may bind stronger than linear genomic DNA), (iii) shift of ligand pK(a) (due to the proximity of highly charged pDNA), and (iv) the solid support itself (steric factors may lead to kinetically stable complexes). The last factor was derived from several comparisons between support-bound ligand and free soluble ligand. It was thus observed that polyelectrolyte complexes associated with a surface were much more difficult to dissociate than the equivalent soluble complexes.     

Negative chromatography: Progress,applications and future perspectives

In negative chromatography, the impurities bind on the adsorbent, and the product is allowed to flow through the chromatographic column. Negative chromatography is an alternative to positive chromatography under certain circumstances and has been used to purify various biomolecules. For this review, a detailed survey of the performance of reported studies on negative chromatography was conducted. The applications of negative chromatography in the capture and intermediate purification steps for biomolecules (e.g., plasmid DNA, antibodies, enzymes, hemoglobin, virus particles and cells) are reviewed. The negative chromatographic adsorbents adsorb the impurities through surface charge, hydrophobic interaction at specific sites on the surface, hydrophobic interaction, hydrogen bonding and functional groups. Examples of applications of negative chromatography according to the type of chromatography matrix used are summarized and discussed. In addition, the effects of operating conditions (initial protein concentration, buffer ions, pH and salt concentration) are discussed, and the criteria for choosing negative or positive chromatography are summarized. The literature survey showed that there will be future limitations and challenges ahead in implementation of negative chromatography. Possible solutions to the limitations and challenges of negative chromatography and future trends for developing negative chromatography are discussed.     

Enzyme purification by hydrophobic chromatography: an alternative approach illustrated in the purification of aspartate transcarbamoylase from wheat germ (Short Communication)

Two adsorbents containing similar numbers of hydrocarbon (C(10)) chains but different numbers of carboxyl groups were made by chemical modification of Sepharose. The use of these adsorbents to purify proteins, under conditions where hydrophobic adsorption is partly resisted by electrostatic repulsion, is illustrated in the purification of aspartate transcarbamoylase (EC 2.1.3.2) from wheat germ.     

Adsorption mechanism at the molecular level between polymers and uremic octapeptide by the 2D 1H NMR Technique

To remove uremic octapeptide from the blood stream of uremic patients, various modified polyacylamide cross-linked absorbents were prepared. Adsorption experiments showed these absorbents have significant differences in adsorption capacity to the target peptide. In this paper, two-dimension proton nuclear magnetic resonance (2D 1H NMR) spectroscopy was used to investigate the interaction mechanism between the peptide and the adsorbents. Because of the insolubility of the absorbent, some soluble linear polymers with the same functional groups as the absorbents were employed as the model adsorbents in 2D 1H NMR. The preferred binding site for the peptide and polymers was identified to be at the C-terminal carboxyl group of the octapeptide via chemical shift perturbation effects. In this study, we found that hydrogen bonding, electrostatic, and hydrophobic interactions all play a role in the interaction force but had different contributions. Especially, the great chemical shift changes of the aromatic amino acid residues (Trp) during the interaction between butyl-modified polyacrylamide and octapeptide suggested the hydrophobic interaction, incorporated with the electrostatic force, played an important role in the binding reaction in aqueous solutions. This information not only rationally explained the results of the adsorption experiments, but also identified the effective binding site and mechanism, and shall provide a structural basis for designing better affinity-type adsorbents for the target peptide.     

Adsorption of phenol, p-chlorophenol and p-nitrophenol onto functional chitosan

Functional chitosan, chemically modified by salicylaldehyde (CS-SA), beta-cyclodextrin (CS-CD), and a cross-linked beta-cyclodextrin polymer (EPI-CD) were prepared as adsorbents to remove phenol, p-nitrophenol and p-chlorophenol from aqueous solution. Langmuir and Freundlich models were applied to describe the adsorption isotherm of phenols, and adsorption parameters were evaluated. Functional chitosan displayed outstanding adsorption ability for phenols. To our surprise, CS-CD exhibited specific adsorption ability for p-chlorophenol. The possible adsorption interaction was discussed. Effects of pH and KCl on the adsorption suggested that the adsorption of phenols was predominated by hydrogen bonding, hydrophobic interaction and pi-pi interaction not electrostatic interaction. Effect of temperature indicated that the low temperature was favorable for the adsorption of phenols. Separation of phenols and adsorbent regeneration were carried out by simple washing with ethanol and filtrating.     

Interaction of inorganic pyrophosphatase from yeast with alkylated derivatives of Sepharose

The adsorption of inorganic pyrophosphatase from baker's yeast by alkylated polysaccharides involves both electrostatic and hydrophobic interactions. The enzyme is eluted by NaCl solutions of different ionic strengths depending on the adsorbent hydrophobicity. The degree of purification on different adsorbents varies from 2- to 80-fold.     

Effects of polyamines on the ultrafiltration of plasmid DNA

It is well established that the structure of plasmid DNA is a strong function of solution ionic conditions due to changes in intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. Multivalent cations like spermine and spermidine play a critical role in compacting and controlling the structure of supercoiled DNA in living cells. The objective of this work was to investigate the effects of these polyamines on the ultrafiltration of plasmid DNA, including possible opportunities to use these polycations to enhance the purification of specific plasmid isoforms. Data were obtained using a wide range of spermine and spermidine concentrations to evaluate DNA transmission through Biomax polyethersulfone ultrafiltration membranes. Spermine has a very strong effect on DNA transmission, with the sieving coefficient of the supercoiled plasmid decreasing by more than an order of magnitude upon addition of only 15 μM spermine. A comparable change in DNA transmission required >300 μM of the trivalent spermidine. The polyamines were able to significantly increase the selectivity for the separation of DNA from a model protein, but they were unable to provide a significant increase in the selectivity for separating DNA isoforms under the conditions examined in this study. The results do demonstrate that both spermine and spermidine can be used to control the extent of DNA transmission/purification during ultrafiltration. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2765, 2019.     

Analysis of the structure of dimeric DNA catenanes by electron microscopy.

We analyzed the structure of open-circular and supercoiled dimeric DNA catenanes generated by site-specific recombination in vitro. Electron microscopy of open-circular catenanes shows that the number of duplex crossings in a plane is a linear function of the number of catenane interlinks (Ca/2), and that the length of the catenane axis is constant, independent of Ca. These relationships are similar to those observed with supercoiled DNA. Statistical analyses reveal, however, that the conformations of the individual rings of the catenanes are similar to those of unlinked circles. The distribution of distances between randomly chosen points on separate rings depends strongly on Ca and is consistent with a sharp decrease in the center-of-mass separation between rings with increasing Ca. Singly linked supercoiled catenanes are seen by microscopy to be linked predominantly through terminal loops in the respective superhelices. The observations suggest that chain entropy is a major factor determining the conformation of DNA catenanes.     

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.     

Thiophilic interaction chromatography for supercoiled plasmid DNA purification

Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.     

Separation of plasmid DNA isoforms using centrifugal ultrafiltration

Centrifugal ultrafiltration is a well-established method for concentrating and purifying DNA. Here, we describe the use of centrifugal ultrafiltration for the separation of plasmid DNA isoforms based on differences in elongational flexibility of the supercoiled, open-circular, and linear plasmids. Transmission of each isoform is minimal below a critical value of the filtration velocity, which is directly related to the magnitude of the centrifugal speed and the system geometry. A discontinuous diafiltration process was used to enrich the desired isoform, as determined by agarose gel electrophoresis. The simplicity and efficacy of this membrane-based separation are attractive for multiple applications requiring the use of separated DNA isoforms.     

Plasmid DNA adsorption on silica: kinetics and conformational changes in monovalent and divalent salts

A quartz crystal microbalance with dissipation (QCM-D) is used to determine the adsorption rate of a supercoiled plasmid DNA onto a quartz surface and the structure of the resulting adsorbed DNA layer. To better understand the DNA adsorption mechanisms and the adsorbed layer physicochemical properties, the QCM-D data are complemented by dynamic light scattering measurements of diffusion coefficients of the DNA molecules as a function of solution ionic composition. The data from simultaneous monitoring of variations in frequency and dissipation energy with the QCM-D suggest that the adsorbed DNA layer is more rigid in the presence of divalent (calcium) cations compared to monovalent (sodium) cations. Adsorption rates are significantly higher in the presence of calcium, attaining a transport-limited rate at about 1 mM Ca2+. Results further suggest that in low ionic strength solutions containing 1 mM Ca2+ and in moderately high ionic strength solutions containing 300 mM NaCl, plasmid DNA adsorption to negatively charged mineral surfaces is irreversible.     

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.     

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.     

Winding of the DNA helix by divalent metal ions.

When supercoiled pBR322 DNA was relaxed at 0 or 22 degrees C by topoisomerase I in the presence of the divalent cations Ca2+, Mn2+ or Co2+, the resulting distributions of topoisomers observed at 22 degrees C had positive supercoils, up to an average delta Lk value of +8.6 (for Ca2+at 0 degrees C), corresponding to an overwinding of the helix by 0.7 degrees/bp. An increase of the divalent cation concentration in the reaction mixture above 50 mM completely reversed the effect. When such ions were present in agarose electrophoresis gels, they caused a relaxation of positively supercoiled DNA molecules, and thus allowed a separation of strongly positively supercoiled topoisomers. The effect of divalent cations on DNA adds a useful tool for the study of DNA topoisomers, for the generation as well as separation of positively supercoiled DNA molecules.     

Effectiveness and mechanisms of dye adsorption on a straw-based biochar

A biochar (BC) generated from straw as a cost-effective substitute for activated carbon (AC) was tested for its adsorptive ability toward reactive brilliant blue (KNR) and rhodamine B (RB). BC and AC had similar surface areas but differed in porosity, surface acidity and point of zero surface charge. The two carbons were highly effective adsorbents for both dyes at pH 3.0 and 6.5. BC was slightly more effective than AC to adsorb RB due to the RB–BC electrostatic interactions and RB protonation at low pH. The two carbons reversed in their effectiveness to adsorb KNR for similar reasons. The π–π interactions between dye molecules and graphene layers of BC, the direct dye-BC electrostatic attraction/repulsion and the intermolecular hydrogen bonding are proposed to be the combined mechanisms for dye adsorption. Rich phenolic hydroxyls on the surface of BC are expected to enhance the π–π interactions.