Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

Kepka, Cecilia; Rhodin, Jenny; Lemmens, Raf; Tjerneld, Folke; Gustavsson, Per-Erik

doi:10.1016/j.chroma.2003.10.028

Cited by 69 publications

(46 citation statements)

References 23 publications

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“…ATPE systems consist of two nearly immiscible aqueous phase‐forming agents that comprise a liquid–liquid partitioning technique. The agents can be two polymer solutions in which the phase separation phenomenon is induced by incompatibility, such as poly(ethylene glycol)‐dextran (PEG‐Dx); a polymer and a lyotropic salt solution with phase separation induced by Coulombic interactions, such as PEG‐potassium phosphate;4 two thermoseparating polymer solutions with phase separation induced by thermal energy, such as poly(ethylene oxide)‐poly(propylene oxide);5 or a pH‐responsive polymer solution with phase separation induced by pH changes, such as poly(diallyamino ethanoate)‐dimethyl sulfoxide 6. One phase‐forming agent will predominate in each phase.…”

Section: Introductionmentioning

confidence: 99%

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

Ahmad

Hauan

Przybycien

2010

J of Chemical Tech & Biotech

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BACKGROUND: Aqueous two-phase extraction (ATPE) has many advantages as an efficient, inexpensive large-scale liquid-liquid extraction technique for protein separation. However, the realization of ATPE as a protein separation technology at industrial scales is rather limited due to the large, multidimensional design space and the paucity of design approaches to predict phase and product behavior in an integrated fashion with overall system performance. This paper describes a framework designed to calculate suitable flowsheets for the extraction of a target protein from a complex protein feed using ATPE. The framework incorporated a routine to set up flowsheets according to target protein partitioning behavior in specific ATPE systems and a calculation of the amounts of phase-forming components needed to extract the target protein. The thermodynamics of phase formation and partitioning were modeled using Flory-Huggins theory and calculated using a Gibbs energy difference minimization approach.

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Section: Introductionmentioning

confidence: 99%

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

Ahmad

Hauan

Przybycien

2010

J of Chemical Tech & Biotech

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“…After electrophoresis, the gel was visualized with a gel documentation system (Vilber Lourmat, Marne-laVallée Cedex 1, France), and the isoforms of pDNA and RNA were identified by bright discrete bands. The contents of pDNA, RNA, and other impurities in the eluted fractions as well as clarified cell lysate were analyzed on a MiniQ 4.6/50 PE column as described by Kepka et al [41]. The column was connected to an Agilent 1100 HPLC system and equilibrated with 50% of buffer B at a flow rate of 0.5 mL/min.…”

Section: Methodsmentioning

confidence: 99%

Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: Effects of spacer arms and ligand density

Bai

Shi

et al. 2014

J. Sep. Science

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Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine-affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10-atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two-stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine-affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate.

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“…Aqueous two-phase extraction (APTE) has also been used to purify cells, viral particles, and plasmid DNA [137–139]. Properties of biomolecules, like hydrophobicity, surface charge, size, and system composition, affect the partitioning of the molecule in any two-phase system.…”

Section: Phase Partitioningmentioning

confidence: 99%

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

Saraswat

Musante

Ravidá

et al. 2013

BioMed Research International

134

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Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

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Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

Cited by 69 publications

References 23 publications

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

Flowsheet simulation of aqueous two‐phase extraction systems for protein purification

Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine-affinity chromatography: Effects of spacer arms and ligand density

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

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