The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate

Almeida, Ana Margarida; Eusébio, Dalinda; Queiroz, João A.; Sousa, Fani

doi:10.1016/j.chroma.2019.460444

Cited by 16 publications

(12 citation statements)

References 19 publications

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“…Protein is stable in PEI and AS precipitation and could be purified to remove much DNA. Furthermore, through the preparation of proper buffers, HIC chromatography, MBP chromatography, and MonoQ chromatography can be performed one by one to save time and also to remove DNA [ 32–37 ]. This strategy results in enough high yield and purity of the target protein.…”

Section: Discussionmentioning

confidence: 99%

Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

2021

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DNA N6-methyladenine (6mA), a kind of DNA epigenetic modification, is widespread in eukaryotes and prokaryotes. An enzyme activity study coupled with 6mA detection using ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) is commonly applied to investigate 6mA potentially related enzymes in vitro. However, the protein expressed in a common Escherichia coli (E. coli) strain shows an extremely high 6mA background due to a minute of co-purified bacterial DNA, though it has been purified to remove DNA using multiple strategies. Furthermore, as occupied by DNA with abundant 6mA, the activity of 6mA related proteins will be influenced seriously. Here, to address this issue, we for the first time construct a derivative of E. coli Rosetta (DE3) via the λRed knockout system specifically for the expression of 6mA related enzymes. The gene dam encoding the 6mA methyltransferase (MTase) is knocked out in the newly constructed strain named LAMBS (low adenine methylation background strain). Contrasting with E. coli Rosetta (DE3), LAMBS shows an ultra-low 6mA background on the genomic DNA when analyzed by UHPLC-MS/MS. We also demonstrate an integral strategy of protein purification, coupled with the application of LAMBS. As a result, the purified protein expressed in LAMBS exhibits an ultra-low 6mA background comparing with the one expressed in E. coli Rosetta (DE3). Our integral strategy of protein expression and purification will benefit the in vitro investigation and application of 6mA related proteins from eukaryotes, although these proteins are elusive until now.

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Section: Discussionmentioning

confidence: 99%

Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

2021

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“…To obtain the mcDNA-E7 vector, a modified alkaline lysis method was performed as described by Diogo et al [ 27 ]. To isolate the mcDNA vector, resulting supernatants were loaded directly onto a Sephacryl SF-1000 instrument, as previously described by our research group [ 28 ]. An AKTA Pure system (GE Healthcare, Buckinghamshire, UK) with UNICORNTM 6.3 software (GE Healthcare, Buckinghamshire, UK) was used to perform all chromatographic runs.…”

Section: Methodsmentioning

confidence: 99%

Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine

Serra

Eusébio²,

Neves³

et al. 2021

Pharmaceutics

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DNA vaccines still represent an emergent area of research, giving rise to continuous progress towards several biomedicine demands. The formulation of delivery systems to specifically target mannose receptors, which are overexpressed on antigen presenting cells (APCs), is considered a suitable strategy to improve the DNA vaccine immunogenicity. The present study developed binary and ternary carriers, based on polyethylenimine (PEI), octa-arginine peptide (R8), and mannose ligands, to specifically deliver a minicircle DNA (mcDNA) vaccine to APCs. Systems were prepared at various nitrogen to phosphate group (N/P) ratios and characterized in terms of their morphology, size, surface charge, and complexation capacity. In vitro studies were conducted to assess the biocompatibility, cell internalization ability, and gene expression of formulated carriers. The high charge density and condensing capacity of both PEI and R8 enhance the interaction with the mcDNA, leading to the formation of smaller particles. The addition of PEI polymer to the R8-mannose/mcDNA binary system reduces the size and increases the zeta potential and system stability. Confocal microscopy studies confirmed intracellular localization of targeting systems, resulting in sustained mcDNA uptake. Furthermore, the efficiency of in vitro transfection can be influenced by the presence of R8-mannose, with great implications for gene expression. R8-mannose/PEI/mcDNA ternary systems can be considered valuable tools to instigate further research, aiming for advances in the DNA vaccine field.

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“…When the sample is applied on the top and passed through the column, smaller molecules such as mRNA and proteins enter through the small pores and channels of the beads, while DNA is excluded from entering the beads and evades the matrix with its larger hydrodynamic volume. Consequently, DNA is eluted from the column faster than the smaller molecules [18,19]. SEC is suitable for use on substances that are sensitive to alterations in pH and metal ion concentrations [20].…”

Section: Methodsmentioning

confidence: 99%

The Evolution of DNA Extraction Methods

Shetty

2020

AJBSR

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Since the first DNA extraction performed by Friedrich Miescher in 1869, scientists have made extraordinary progress in designing extraction methods that are more reliable, easier and faster to perform, more cost-effective and produce a higher yield. The classic liquid-liquid DNA extraction method involves the use of organic and inorganic reagents such as phenol-chloroform which pose a toxic threat to humans. Many newer techniques are now based on physical extraction, which has significantly contributed to developing simpler methods for DNA handling, such as extraction using magnetic beads and cellulose-based filter paper. With the advent of gene-editing and personalized medicine, there has been an increase in the demand for reliable and efficient DNA isolation methods that can yield adequate quantities of high-quality DNA with minimal impurities. The current review addresses the evolution of different DNA extraction techniques from solvent-based methods to physical extraction methods each with its varying set of advantages and limitations.

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The use of size-exclusion chromatography in the isolation of supercoiled minicircle DNA from Escherichia coli lysate

Cited by 16 publications

References 19 publications

Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N6-methyladenine backgrounds in Escherichia coli

Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine

The Evolution of DNA Extraction Methods

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