Plasmid DNA purification using a multimodal chromatography resin

Multimodal chromatography is widely used for isolation of proteins because it often results in improved selectivity compared to conventional separation resins. The binding potential and chromatographic behavior of plasmid DNA have here been examined on a Capto Adhere resin. Capto Adhere is a recent multimodal chromatography material allowing molecular recognition between the ligand and target molecule, which is based on combined ionic and aromatic interactions. Capto Adhere proved to offer a very strong binding of nucleic acids. This property could be used to isolate plasmid DNA from a crude Escherichia coli extract. Using a stepwise NaCl gradient, pure plasmid DNA could be obtained without protein and endotoxin contaminations. The RNA fraction bound most strongly to the resin and could be eluted only at very high salt concentrations (2.0 M NaCl). The chromatographic separation behavior was very robust between pH values 6 and 9, and the dynamic binding capacity was estimated to 60 µg/ml resin. Copyright © 2014 John Wiley & Sons, Ltd.     

Purification of a cystic fibrosis plasmid vector for gene therapy using hydrophobic interaction chromatography

The success and validity of gene therapy and DNA vaccination in in vivo experiments and human clinical trials depend on the ability to produce large amounts of plasmid DNA according to defined specifications. A new method is described for the purification of a cystic fibrosis plasmid vector (pCF1-CFTR) of clinical grade, which includes an ammonium sulfate precipitation followed by hydrophobic interaction chromatography (HIC) using a Sepharose gel derivatized with 1,4-butanediol-diglycidylether. The use of HIC took advantage of the more hydrophobic character of single-stranded nucleic acid impurities as compared with double-stranded plasmid DNA. RNA, denatured genomic and plasmid DNAs, with large stretches of single strands, and lipopolysaccharides (LPS) that are more hydrophobic than supercoiled plasmid, were retained and separated from nonbinding plasmid DNA in a 14-cm HIC column. Anion-exchange HPLC analysis proved that >70% of the loaded plasmid was recovered after HIC. RNA and denatured plasmid in the final plasmid preparation were undetectable by agarose electrophoresis. Other impurities, such as host genomic DNA and LPS, were reduced to residual values with the HIC column (<6 ng/microg pDNA and 0.048 EU/microg pDNA, respectively). The total reduction in LPS load in the combined ammonium acetate precipitation and HIC was 400,000-fold. Host proteins were not detected in the final preparation by bicinchoninic acid (BCA) assay and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Plasmid identity was confirmed by restriction analysis and biological activity by transformation experiments. The process presented constitutes an advance over existing methodologies, is scaleable, and meets quality standards because it does not require the use of additives that usually pose a challenge to validation and raise regulatory concerns.     

Metal chelate affinity precipitation of RNA and purification of plasmid DNA

The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine `tails'. We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines. Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA. A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E. coli. The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands. RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl. Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step. RNA binding showed a strong dependence on temperature and on the type of buffer used.     

Quantitation of plasmid DNA in aqueous two-phase systems by fluorescence analysis

The development of aqueous two-phase systems for plasmid purification from Escherichia coli cell lysates requires a reliable DNA quantitation method. Plasmid DNA was quantified by fluorescence using PicoGreen nucleic acid stain. Linearity was obtained up to 40 ng plasmid ml^–1. Two polyethyleneglycol (PEG)/salt systems were studied, PEG 600/K₂HPO₄ and PEG 300/K₂HPO₄. The average plasmid recovery was 41% in the bottom phase of the first system and 35% in the top phase of the second system. This method has proved to be simple and reproducible.     

In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida*†

SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH₄)₂SO₄ and 0.13M (NH₄)₂SO₄ respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn²⁺. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.     

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).     

Chromatographic Separation, and Characteristics of Nucleic Acids from HeLa Cells

The application of the phenol-duponol method to extraction of nucleic acids from HeLa cells is described. Chromatography of the phenol extract on an esterified bovine serum albumin column with a salt gradient of sodium chloride gives separation of soluble RNA, DNA, and two different high molecular RNA fractions. Ultracentrifugation of the DNA eluted from the column gives a sedimentation coefficient (s₂₀^o,w) of 38, which agrees with ultracentrifugation data on the phenol extract. The eluted RNA appears polydisperse at low ionic strength, but at high ionic strength and after alcohol precipitation two fractions with the sedimentation coefficients of 16 and 25 to 29, respectively, were obtained.     

Direction of transcription affects the replication mode of lambda in an in vitro system

Summary pMV158 is a 5.4 kb broad host range multicopy plasmid specifying tetracycline resistance. This plasmid and two of its derivatives, pLS1 and pLS5, are stably mantained and express their genetic information in gram-positive and gram-negative hosts. The in vitro replication of plasmid pMV158 and its derivatives was studied in extracts prepared from plasmid-free Escherichia coli cells and the replicative characteristics of the streptococcal plasmids were compared to those of the E. coli replicons, ColE1 and the mini-R1 derivative pKN182. The optimal replicative activity of the E. coli extracts was found at a cellular phase of growth that corresponded to 2 g wet weight of cells per litre. Maximal synthesis of streptococcal plasmid DNA occurred after 90 min of incubation and at a temperature of 30° C. The optimal concentration of template DNA was 40 g/ml. Higher plasmid DNA concentrations resulted in a decrease in the incorporation of dTMP, indicating that competition of specific replication factor(s) for functional plasmid origins may occur. In vitro replication of plasmid pMV158 and its serivatives required the host RNA polymerase and de novo protein synthesis. The final products of the streptococcal plasmid DNAs replicated in the E. coli in vitro system were monomeric supercoiled DNA forms that had completed at least one round of replication, although a set of putative replicative intermediates could also be found. The results suggest that a specific plasmid-encoded factor is needed for the replication of the streptococcal plasmids.     

Tris-chelated complexes of nickel(II) with bipyridine derivatives: DNA binding and cleavage,BSA binding,molecular docking,and cytotoxicity

Abstract

Two nickel(II) complexes with substituted bipyridine ligand of the type [Ni(NN)₃](ClO₄)₂, where NN is 4,4′-dimethyl-2,2′-bipyridine (dimethylbpy) (1) and 4,4′-dimethoxy-2,2′-bipyridine (dimethoxybpy) (2), have been synthesized, characterized, and their interaction with DNA and bovine serum albumin (BSA) studied by different physical methods. X-ray crystal structure of 1 shows a six-coordinate complex in a distorted octahedral geometry. DNA-binding studies of 1 and 2 reveal that both complexes sit in DNA groove and then interact with neighboring nucleotides differently; 2 undergoes a partial intercalation. This is supported by molecular-docking studies, where hydrophobic interactions are apparent between 1 and DNA as compared to hydrogen bonding, hydrophobic, and π–π interactions between 2 and DNA minor groove. Moreover, the two complexes exhibit oxidative cleavage of supercoiled plasmid DNA in the presence of hydrogen peroxide as an activator in the order of 1?>?2. In terms of interaction with BSA, the results of spectroscopic methods and molecular docking show that 1 binds with BSA only via hydrophobic contacts while 2 interacts through hydrophobic and hydrogen bonding. It has been extensively demonstrated that the nature of the methyl- and methoxy-groups in ligands is a strong determinant of the bioactivity of nickel(II) complexes. This may justify the above differences in biomolecular interactions. In addition, the in vitro cytotoxicity of the complexes on human carcinoma cells lines (MCF-7, HT-29, and U-87) has been examined by MTT assay. According to our observations, 1 and 2 display cytotoxicity activity against selected cell lines.

Communicated by Ramaswamy H. Sarma     

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.     

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein ¹. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH₄)₂SO₄). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) ². As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter ³. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs ⁴.The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light ⁵. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.Download video file.^{(63M, mov)}     

Purification of plasmid DNA using a multicompartment electrolyser separated by ultrafilter membranes

A simple, scalable method for purification of plasmid DNA is described. Plasmid DNA was released from Escherichia coli JM109 by lysis (1% SDS, 0.2 M NaOH). Then a neutralization solution (3 M sodium acetate buffer, pH 4.8) was added to precipitate genomic DNA and protein. After the clarification of the lysate, the supernatant was placed in a multicompartment electrolyser separated by ultrafilter membranes to remove the remaining contamination (RNA, genomic DNA and protein). A recovery of 75%±2% of total plasmid DNA was obtained after 60 min electrophoresis with a field strength of 8 V cm^–1 using cells at 30 g l^–1 (quantified by dry cell weight). Genomic DNA, RNA and protein were undetectable in the purified plasmid DNA solution.     

Time-course determination of plasmid content in eukaryotic and prokaryotic cells using Real-Time PCR

A Real-Time PCR method was developed to monitor the plasmid copy number (PCN) in Escherichia coli and Chinese hamster ovary (CHO) cells. E. coli was transformed with plasmids containing a ColE1 or p15A origin of replication and CHO cells were transfected with a ColE1 derived plasmid used in DNA vaccination and carrying the green fluorescent protein (GFP) reporter gene. The procedure requires neither specific cell lysis nor DNA purification and can be performed in <30 min with dynamic ranges covering 0.9 pg–55 ng, and 5.0 pg–2.5 ng of plasmid DNA (pDNA) for E. coli and CHO cells, respectively. Analysis of PCN in E. coli batch cultures revealed that the maximum copy number per cell is attained in mid-exponential phase and that this number decreases on average 80% towards the end of cultivation for both types of plasmids. The plasmid content of CHO cells determined 24 h post-transfection was around 3 × 10⁴ copies per cell although only 37% of the cells expressed GFP one day after transfection. The half-life of pDNA was 20 h and around 100 copies/cell were still detected 6 days after transfection.     

RNase-sensitive DNA polymerase activity in cell fractions and mutants of Neurospora crassa

RNase-sensitive DNA polymerase activity (RSDP) was tested in different cell fractions of Neurospora crassa cell types and its morphological mutants. This RSDP was found localized in the microsomal pellet fraction and absent in the purified nuclear pellets isolated from different N. crassa cell types: conidia, germinated conidia, and mycelia. This enzyme is capable of synthesizing a DNA product only in the presence of all four deoxyribonucleoside-5-triphosphates and Mg²⁺. Removal of RNA from the pellet fraction by RNase strongly inhibited the DNA synthesis. The endogenous synthesis of DNA in the microsomal pellet fraction was associated with the formation of an RNA:DNA hybrid as analyzed by Cs₂SO₄ equilibrium density gradient centrifugation. The DNA product after alkali hydrolysis hybridizes with the RNA isolated from the same pellet fraction, as analyzed by elution from hydroxylapatite column at 60 C. This DNA product did not hybridize with poly(A). A few mutants tested showed this RNase-sensitive DNA polymerase activity.This work was supported in part by a contract with the U.S. Department of Energy and a grant from the U.S. Naval Research.     

Characterization of adhesion associated surface properties of uropathogenicEscherichia coli

Escherichia coli was isolated from the urine of patients with pyelonephritis, with urinary tract infections other than pyelonephritis and with asymptomatic bacteriuria. Surface properties of the strains were analyzed by the salting-out aggregation test (SAT), hydrophobic interaction chromatography (HIC), Congo red binding (Crb), agglutination of erythrocytes (MRHA) and latex particles covered by digalactoside (PF) and by adherence to tissue culture cells. In addition, a DNA probe for thepap gene was used. The DNA probe detected the highest proportion of strains withpap gene in the group of patients with pyelonephritis, lower in the urinary tract infections other than pyelonephritis and the lowest in the group with asymptomatic bacteriuria. Tests for P-fimbriae (PF, MRHA) showed a similar distribution. Hydrophobicity measured by SAT and by HIC did not show differences among the tested groups of strains. The results suggest that factors other than the P-fimbriae and hydrophobicity may contribute to the persistence ofE. coli in the urinary tract.     

Overexpression of the gene forN-acylamino acid racemase fromAmycolatopsis sp. TS-1-60 inEscherichia coli and continuous production of optically active methionine by a bioreactor

A DNA sequence encodingN-acylamino acid racemase (AAR) was inserted downstream from the T7 promoter in pET3c. The recombinant plasmid was introduced intoEscherichia coli MM194 lysogenized with a bacteriophage having a T7 RNA polymerase gene. The amount of AAR produced by theE. coli transformant was 1100-fold more than that produced byAmycolatopsis sp. TS-1-60, the DNA donor strain. The AAR was purified to homogeneity from the crude extract of theE. coli transformant by two steps: heat treatment and Butyl-Toyopearl column chromatography. Bioreactors for the production of optically active amino acids were constructed with DEAE-Toyopearl-immobilized AAR andd- orl-aminoacylase.d- orl-methionine was continuously produced with a high yield fromN-acetyl-dc-methionine by the bioreactor.     

Escherichia coli and Pseudomonas putida RNA polymerases display identical contacts with promoters

Summary Methylation protection experiments with four promoters (P1 and P2 of the pBR322 plasmid, lacUV5 and lambda P₀) have shown that the RNA polymerases from Escherichia coli and Pseudomonas putida, while differing in the primary structure of the subunits involved in DNA binding, display identical patterns of DNA contacts. Nor do these enzymes differ in covalent cross-linking patterns with a partially apurinized promoter. We conclude that the two RNA polymerases have very similar structures of DNA binding centers. The evolutionary conservation of this structure may account for the fact that diverse RNA polymerases often recognize and efficiently use promoters of distant bacterial species.     

Isolation of genomic DNA suitable for community analysis from mature trees adapted to arid environment

Isolation of intact and pure genomic DNA (gDNA) is essential for many molecular biology applications. It is difficult to isolate pure DNA from mature trees of hot and dry desert regions because of the accumulation of high level of polysaccharides, phenolic compounds, tannins etc. We hereby report the standardized protocol for the isolation and purification of gDNA from seven ecologically and medically important tree species of Combretaceae viz. Anogeissus (Anogeissus sericea var. nummularia, Anogeissus pendula, and Anogeissus latifolia) and Terminalia (Terminalia arjuna, Terminalia bellirica, Terminalia catappa and Terminalia chebula). This method involves (i) washing the sample twice with Triton buffer (2%) then (ii) isolation of gDNA by modified-CTAB (cetyl trimethyl ammonium bromide) method employing a high concentration (4%) of PVP (Polyvinylpyrrolidone) and 50 mM ascorbic acid, and (iii) purification of this CTAB-isolated gDNA by spin-column. gDNA isolated by modified CTAB or spin-column alone were not found suitable for PCR amplification. The Triton washing step is also critical. The quality of DNA was determined by the A₂₆₀/A₂₈₀ absorbance ratio. gDNA was also observed for its intactness by running on 0.8% agarose gel. The suitability of extracted DNA for PCR was tested by amplification with RAPD primers, which was successful. Further, rbcLa (barcoding gene) was amplified and sequenced to check the quality of extracted gDNA for its downstream applications.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

A new eukaryotic RNA polymerase factor: a factor from chicken myeloblastosis cells which stimulates transcription of denatured DNA

A heat-stable protein factor, capable of stimulating RNA synthesis by nuclear RNA polymerase II, was found in isolated nuclei of chicken myeloblastosis cells. It is adsorbed to a DEAE-Sephadex column used for RNA polymerase purification and then is eluted with 0.1 M ammonium sulfate. This factor appears to differ from previously reported eukaryotic RNA polymerase factors in its property of stimulating the activity of denatured (or single-stranded) DNA template. When heated, this factor contains no detectable endonuclease or exonuclease activity. The degree of stimulation is greater with chicken myeloblastosis RNA polymerase IIb than IIa and is most efficient when homologous DNA is used as template. This factor causes no stimulation of $\text{E. coli}$ RNA polymerase.