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WO2010046918A1 - Purification d’adn de plasmide superenroulé - Google Patents

Purification d’adn de plasmide superenroulé Download PDF

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Publication number
WO2010046918A1
WO2010046918A1 PCT/IN2009/000558 IN2009000558W WO2010046918A1 WO 2010046918 A1 WO2010046918 A1 WO 2010046918A1 IN 2009000558 W IN2009000558 W IN 2009000558W WO 2010046918 A1 WO2010046918 A1 WO 2010046918A1
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WO
WIPO (PCT)
Prior art keywords
dna
supercoiled
plasmid
plasmid dna
metal ions
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Ceased
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PCT/IN2009/000558
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English (en)
Inventor
Munishwar Nath Gupta
Pradeep K. Singh
Smita Raghava
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VB Medicare Pvt Ltd
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VB Medicare Pvt Ltd
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Publication date
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Publication of WO2010046918A1 publication Critical patent/WO2010046918A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the invention relates to isolation and purification of ScDNA (supercoiled plasmid DNA) for the purpose of use as a vaccine.
  • DNA vaccines have come up as a very effective tool for immunization that has several advantages over conventional vaccines.
  • the concept comprises introducing a gene of a pathogenic organism in a host in a way that facilitates expression of an antigen in the host organism that will trigger a response from the host organism for production of antibodies against that antigen and effectively against the organism from which the gene is derived.
  • plasmids in which the desired pathogen gene is incorporated, transforming a microbe, such as Escherichia coli, large scale fermentative production of the transformed microbe, stimulating over expression of the plasmids containing the pathogen gene construct as overexpressed inclusion bodies in the form of super-coiled plasmids in the transformed microbe, recovering the super-coiled plasmids thus formed and using them as an active ingredient of a vaccine alone or in combination with other supercoiled plasmids.
  • a microbe such as Escherichia coli
  • large scale fermentative production of the transformed microbe stimulating over expression of the plasmids containing the pathogen gene construct as overexpressed inclusion bodies in the form of super-coiled plasmids in the transformed microbe, recovering the super-coiled plasmids thus formed and using them as an active ingredient of a vaccine alone or in combination with other supercoiled plasmids.
  • Several advantages of such a vaccine include stability of this vaccine at room temperature, ability to make cocktail of several ScDNA species, ability of these vaccines to stimulate both antibody production and cytotoxic T-cell response and good safety profile as toxicity/immunogenicity is low.
  • Such a vaccine can not afford to have various contaminants from other cell components as impurities, particularly plasmid isoforms (linear, relaxed), genomic DNA and RNA of the transformed cell, proteins and endotoxins from the host cell, particularly from E. coli.
  • This invention comprises a process of purifying supercoiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in Gene Therapy or in uses where high purity supercoiled plasmid DNA is required, from a boiling cell lysate or from a solution in water of a mixture comprising supercoiled plasmid DNA and an impurity, the said process comprising steps of treating the said solution by selective precipitation of all DNA and endotoxins except supercoiled DNA by free metal ions in a solution for a period of time, separating supernatant from the precipitate and removing metal ions from the said supernatant.
  • the said impurity comprising plasmid isoforms (linear, relaxed), genomic DNA, RNA, and an endotoxin from a host cell.
  • the method is illustrated by using Cu 2+ metal ions.
  • the said Cu 2+ metal ions were added as CuSO 4 to reach to a final concentration of at least 0.5 M for a period of time of at least about one hour.
  • the separation of supernatant from the precipitate may be achieved by centrifugation.
  • the removal of metal ions is achieved by dialysis The dialysis is done, preferably, against a buffer containing EDTA.
  • This invention is illustrated by isolation of supercoiled plasmid DNA from a genetically transformed bacterium Escherichia coli.
  • the invention can be applied to supercoiled DNA isolation from water solution of over-expressed supercoiled DNA from any genetically modified micro-organism.
  • EU Endotoxin Unit
  • the said boiling cell lysate is prepared comprising steps of centrifuging culture broth to get a pellet of cells, suspending the said pellet in small quantity of buffer and adding Lysozyme to it, putting on boiling water bath for a short time.
  • a process of purifying super-coiled plasmid DNA of purity and characteristics required for use in a DNA vaccine or in a Gene Therapy or in uses where high purity supercoiled DNA is required, from a genetically engineered Escherichia coli containing over- expressed supercoiled DNA comprising steps of centrifuging the culture broth to get a pellet of cells, suspending the said pellet in small quantity of buffer, adding an enzyme to lyse the cells to the same, putting on boiling water bath for a short time to achieve lysis, centrifuging the lysed mixture to remove precipitate comprising proteins and DNA from a supernatant that contains native plasmid DNA and other impurities, adding sodium acetate and isopropanol to the said supenatant, centrifuging to get a pellet of DNA and separating supernatant containing impurities, washing the pellet with 70% ethanol accompanied by centrifugation and dissolving in sterile distilled water to get a solution containing all forms of DNA and end
  • EU Endotoxin Unit
  • the present invention discloses a technology for obtaining supercoiled plasmids with the following characteristics/purity that meet the requirements of the regulatory agencies for the use of such preparations for DNA vaccines/gene therapy (Ferreira et al., 2000; Prather et al., 2003).
  • Prior art methods for recovering super coiled plasmid comprise column chromatography on silica gel, anion exchangers, hydrophobic interactions, chromatographic media. Partition methods have also been explored for large scale methods. Centrifugation in CsCl-ethidium bromide gradient is also used at lab scale.
  • IMAC immobilized metal-ion affinity chromatography
  • IMAC is a chromatographic technique which utilizes chelated metal ions (Cu 2+ , Zn 2+ , Ni 2+ , Co 2+ , Fe 3+ ) and has been more extensively applied for protein purification (Porath et. al., 1975; Porath, 1992).
  • Murphy et. al. (2003) have purified plasmid DNA from E. coli alkaline lysates ,wherein the IMAC matrixes adsorbed single-stranded nucleic acids through metal ion interactions with aromatic base nitrogen.
  • RNA and single-stranded oligonucleotides bind strongly to matrixes such as Cu(II) iminodiacetic acid (IDA) agarose. It has been reported that RNA and damaged plasmid DNA are effectively removed without loss of the closed circular plasmid, presumably through interactions with exposed, single-stranded regions.
  • matrixes such as Cu(II) iminodiacetic acid (IDA) agarose.
  • Tan et. al. (2007) have talked of the differential interactions of plasmid DNA, RNA, and endotoxins with immobilized and free metal ions. Tan et. al., who worked with alkaline cell lysate on immobilized metal ion systems, however, never worked with a real system / cell lysates with free metal ions and it is not possible to simulate/extrapolate their results.
  • the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution.
  • the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from cell lysate of bacteria containing the said supercoiled DNA.
  • the invention comprises use of free metal ions for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from boiling cell lysate of bacteria containing the said supercoiled DNA.
  • the invention comprises use of free Cu 2+ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution.
  • the invention comprises use of free Cu 2+ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from cell lysate of bacteria containing the said supercoiled DNA.
  • the invention comprises use of free Cu 2+ for getting supercoiled plasmids isolated in a form free from other forms of DNA from a mixture of DNA dissolved in distilled water solution; the said solution in distilled water being derived from boiling cell lysate of bacteria containing the said supercoiled DNA.
  • a final concentration of 0.5 M with respect to CuSO 4 gives Cu 2+ concentration that gives precipitation of endotoxin and plasmid isoforms and RNA in the embodiments of the invention mentioned above.
  • treatment with Cu 2+ in above embodiments is carried out for about one hour.
  • treatment with Cu 2+ in above embodiments is carried out for about one hour and at 25°C.
  • this invention comprises a method of obtaining supercoiled DNA preparation useful for DNA Vaccines and gene therapy, and other uses where highly pure supercoiled DNA is required.
  • the vaccines and products produced from supercoiled DNA prepared by the method of this invention are also embodiments of this invention.
  • the invention deals with obtaining the pure supercoiled plasmids with above specifications by the following procedure: A. Cell lysis and primary isolation step. This followed the boiling lysis method as essentially described by Holmes and Quigley (1981). An alternative method called alkaline lysis method (Birnboim and DoIy, 1979) is also available for cell lysis (in case of bacteria) and in fact is often recommended (Brown, 1998; Alcamo, 1999) MNG for plasmid recovery. However, it was surprising that alkaline lysate failed to give purification of supercoiled DNA at 0.5 M concentration of CuSO 4 by the method of this invention; see Fig. 2 lane 4.
  • Steps followed in Figure 1 are in general as follows: Culture broth is centrifuged to get a pellet of cells that is suspended in small quantity of buffer. Lysozyme is added to it and the sample is put on boiling water bath for a short time.
  • the lysed mixture is centrifuged to remove precipitate of proteins and DNA from a supernatant that contains native plasmid DNA and other impurities to which sodium acetate and isopropanol is added and centrifuged to get a pellet of DNA and impurities get separated in supernatant.
  • the pellet is washed with 70% ethanol accompanied by centrifugation and dissolved in sterile distilled water. This solution contained all forms of DNA and endotoxin.
  • Figure 1 protocol for isolation and purification of supercoiled DNA
  • Figure 2 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5 ⁇ harboring pGFPuv plasmid DNA by method described in this application and the one described by Tan et. al., 2007.
  • Lane 1 total nucleic acid prepared by boiling lysis method; Lane 2, after metal precipitation (0.5M Cu2+) of boiling lysate; Lane 3, total nucleic acid prepared by alkaline lysis method (Tan et. al., 2007 ); Lane 4, after metal precipitation (0.5M Cu 2+ ) of alkaline lysate.
  • Figure 3 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5 ⁇ harboring pGFPuv at different concentration of Cu 2+ .
  • Lane 1 total nucleic acid prepared by boiling lysis method; Lane 2, at 0.1 M Cu 2+ ; Lane 3, at 0.25M Cu 2+ ; Lane 4, at 0.5M Cu 2+ ; Lane 5, at 0.75M Cu 2+ ; Lane 6, at 1.0M Cu 2+ .
  • Figure 4 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5 ⁇ harboring pTYB12 containing lipase gene. Lane 1, total nucleic acid (precipitated cell lysate by isopropanol/sodium acetate); Lane 2, precipitate containing RNA and other impurities; Lane 3, supercoiled plasmid DNA obtained by our method.
  • Figure 4a CD spectra of purified supercoiled plasmid DNA, pTYB12 containing lipase gene (1) and total nucleic acid precipitated with isopropanol/ sodium acetate (2) in 10 mM Tris-HCl, ImM EDTA, pH 7.0, 25°C. Concentration of purified supercoiled plasmid DNA was 0.05 mg/mL and 0.06 mg/mL for total nucleic acid.
  • Figure 4b Variation with temperature of UV absorption (245 nm) of purified supercoiled plasmid, pTYB12 containing lipase gene (0.06 mg/mL) in 10 mM Tris-HCl, ImM EDTA, and pH 7.0.
  • Figure 4c Transmission electron micrograph of purified supercoiled plasmid DNA, pTYB12 containing lipase gene, at 50 nm resolution.
  • Figure 4d Comparison of yield of supercoiled plasmid DNA, pTYB12 containing lipase gene, purified by copper precipitation (grey) with Qiagen plasmid purification spin column (black) [QIAprep® Spin Miniprep Kit].
  • Figure 5 0. 7% agarose gel stained with ethidium bromide showing purification of supercoiled plasmid DNA from E. coli DH5 ⁇ harboring pGEM ® T-Easy. Lane 1, total nucleic acid (precipitated cell lysate by isopropanol/sodium acetate); Lane 2, supercoiled plasmid DNA obtained by our method.
  • PLASMID pGFPuv CLONTECH LABORATORIES, PALO ALTO, CA
  • Green fluorescent protein has become well established as a marker of gene expression and protein targeting in intact cells and organisms (Tsien, 1998). The potential of GFP has also been recognized by gene therapy researchers (Wahlfors et. al., 2001).
  • the plasmid pGFPuv was transformed into E. coli DH5 ⁇ . A single colony was picked and inoculated into 5 mL Luria-Bertani (LB) medium containing 100 ⁇ gmL "1 ampicillin. The tube was incubated at 37 0 C at 200 rpm for 14-16 h till an ⁇ 6OO of approximately 4 was reached. The protocol described in Figure 1 was followed thereafter for getting a purified ScDNA preparation.
  • LB Luria-Bertani
  • Figure 3 shows the agarose gel for the purification of ScDNA at different concentration of Cu 2+ . It was observed that 0.5 M Cu 2+ (lane 4, figure 3) was sufficient for getting ScDNA at a purity level in conformity with the criteria stated in the beginning of this application.
  • the plasmid pTYB12 containing lipase gene was transformed into E. coli DH5 ⁇ .
  • a single colony was picked and inoculated into 5 mL Luria-Bertani (LB) medium containing 100 ⁇ gmL 1 ampicillin.
  • the tube was incubated at 37 °C at 200 rpm for 14-16 h till an ⁇ 600 of approximately 4 was reached.
  • the protocol described in Fig 1 was followed thereafter for getting a purified ScDNA preparation.
  • Figure 4 shows the agarose gel for the purification of ScDNA at a purity level in conformity with the criteria stated in the beginning of this application.
  • Figure 4a and 4b (CD spectra and UV melt profile) strongly support that the preparation obtained was of ScDNA (Thumm et.
  • Figure 4c shows the transmission electron micrograph of the final purified preparation.
  • Figure 4d shows the comparison of yield of ScDNA purified by Cu 2+ precipitation with Qiagen plasmid purification spin columns. It is shown that at large culture volumes, the yield obtained by Cu 2+ precipitation was better than what was obtained by using the Qiagen kit.
  • GFP Green fluorescent protein

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Abstract

La présente invention concerne un procédé de purification d’ADN de plasmide superenroulé à un niveau de pureté et de caractéristiques requis pour être utilisé dans un vaccin d’ADN ou une thérapie génique, ou dans des applications nécessitant un ADN de plasmide superenroulé de grande pureté, la purification se faisant à partir d’une bactérie génétiquement modifiée surexprimant ledit plasmide superenroulé. L’invention concerne également l’ADN de plasmide superenroulé préparé par un procédé selon cette invention et des produits contenant un ADN superenroulé produit par un procédé de cette invention. Le procédé selon la présente invention comprend la précipitation sélective, à partir d’un lysat cellulaire, de tous les ADN et de toutes les endotoxines, à l’exception d’ADN superenroulé, par des ions métalliques libres. Ladite impureté comporte des isoformes de plasmide (linéaires, de type relâché), d’ADN et d’ARN génomiques, et une endotoxine d’une cellule hôte. Le procédé est illustré en faisant intervenir des ions métalliques Cu2+. Lesdits ions métalliques Cu2+ ont été ajoutés sous forme de CuSO4 pour obtenir une concentration finale d’au moins 0,5 M. L’élimination des ion métalliques est obtenue par dialyse.
PCT/IN2009/000558 2008-10-08 2009-10-08 Purification d’adn de plasmide superenroulé Ceased WO2010046918A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999016869A1 (fr) * 1997-09-30 1999-04-08 Amersham Pharmacia Biotech Ab Procede de purification d'adn plasmidique
WO2002083893A2 (fr) * 2001-04-18 2002-10-24 Amersham Biosciences Ab Isolement de molecules d'adn
WO2009025690A2 (fr) * 2007-05-23 2009-02-26 Nature Technology Corporation Production améliorée d'adn plasmidique de e.coli

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999016869A1 (fr) * 1997-09-30 1999-04-08 Amersham Pharmacia Biotech Ab Procede de purification d'adn plasmidique
WO2002083893A2 (fr) * 2001-04-18 2002-10-24 Amersham Biosciences Ab Isolement de molecules d'adn
WO2009025690A2 (fr) * 2007-05-23 2009-02-26 Nature Technology Corporation Production améliorée d'adn plasmidique de e.coli

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LEMMENS R. ET AL: "Supercoiled plasmid DNA: selective purification by thiophilic/aromatic adsorption", JOURNAL OF CHROMATOGRAPHY B, vol. 784, 2003, pages 291 - 300 *
PRAZERES D.M.F. ET AL: "Purification of plasmids for gene therapy and DNA vaccination", BIOTECHNOLOGY ANNUAL REVIEW, vol. 7, 2001, pages 1 - 30 *
SAMBROOK J. ET AL: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS, ISBN: 0-87969-309-6, pages: 1.25 - 1.30 *

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