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WO2001029209A1 - Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique - Google Patents

Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique Download PDF

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Publication number
WO2001029209A1
WO2001029209A1 PCT/GB2000/004006 GB0004006W WO0129209A1 WO 2001029209 A1 WO2001029209 A1 WO 2001029209A1 GB 0004006 W GB0004006 W GB 0004006W WO 0129209 A1 WO0129209 A1 WO 0129209A1
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Prior art keywords
restriction enzyme
cellular component
cells
purified
dna
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PCT/GB2000/004006
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English (en)
Inventor
Julian Alexis John Hanak
John Maxim Ward
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Cobra Therapeutics Ltd
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Publication date
Application filed by Cobra Therapeutics Ltd filed Critical Cobra Therapeutics Ltd
Priority to KR1020027004874A priority Critical patent/KR20020064884A/ko
Priority to JP2001532192A priority patent/JP2003512054A/ja
Priority to EP00971521A priority patent/EP1222260A1/fr
Priority to AU10361/01A priority patent/AU1036101A/en
Priority to CA002386401A priority patent/CA2386401A1/fr
Publication of WO2001029209A1 publication Critical patent/WO2001029209A1/fr
Priority to NO20021782A priority patent/NO20021782L/no
Priority to HK02105760.5A priority patent/HK1045539A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • the present invention relates to a method of preparing cellular components that are substantially free of chromosomal DNA.
  • the present invention also relates to host cells for use in the method and cellular components obtained by the method.
  • Restriction enzymes are well known and widely used in the field of recombinant DNA technology. They comprise a group of bacterial endonuclease enzymes that hydrolyse phosphodiester bonds in double-stranded DNA, resulting in cleavage of the DNA at this point.
  • the type II restriction enzymes cleave DNA by this mechanism in a site-specific manner, with each enzyme having a particular 4—12 base recognition sequence (commonly 6), within or adjacent to which they cleave. This process is known as 'restriction'.
  • this cleavage may be prevented by the prior methylation of a particular cytosme or adenine base within the recognition sequence by a corresponding sequence-specific methylase enzyme.
  • This process is known as 'modification'.
  • the biological purpose of this restriction-modification system may be to protect bacteria from the introduction of foreign, viral DNA, not methylated in the appropriate sequences, by bacteriophages.
  • Chromosomal DNA is a major component of cell lysates and considerably hampers purification of cellular components, for example plasmid DNA.
  • the standard method for removal of chromosomal DNA following cell lysis is by denaturation at high pH followed by centrifugation in the well-known 'alkaline lysis' method, or variations thereof (Birnboim and Doly 1979; Sambrook et al. 1989; and European patent application EP-A- 0880585).
  • this is a complex and costly process.
  • Plasmids encoding restriction enzymes and methylases are known in the art for the purpose of positively selecting recombinant plasmids into which DNA inserts have been successfully introduced (O'Connor and Humphreys 1982; Kuhn et al. 1986; Hill et al. 1989).
  • Such positive-selection cloning vectors use the principle that the DNA fragment inserted prevents the expression of a restriction enzyme, that, under suitable conditions, would result in the cleavage and destruction of the plasmid.
  • 'Empty' plasmids are therefore destroyed, increasing the efficiency of the selection of plasmids carrying inserts.
  • Such plasmids have not been used in a method of removing chromosomal DNA during a process of purification of plasmids or other cellular components.
  • a method of removing chromosomal DNA by means of cleavage of the DNA and subsequent digestion by exonucleases, with survival of plasmid DNA is known in the art (Sancar et al. 1979).
  • ultraviolet irradiation is used to generate lesions in the DNA.
  • a strain of cell carrying recA, uvrA mutations is used, so that such lesions are not repaired, but exonucleases degrade the resulting fragments of chromosomal DNA.
  • Plasmid DNA preferentially survives due to the lower probability of UN-generated lesions occurring on the smaller plasmid D ⁇ A molecules.
  • the purpose of this so-called "maxicell” technique is to identify plasmid-encoded, radiolabelled proteins in cell extracts.
  • the present invention provides a method of purifying a cellular component substantially free of chromosomal D ⁇ A comprising culturing cells producing the cellular component in a medium and then exposing the chromosomal D ⁇ A of the cells to a restriction enzyme such that it is cleaved, whilst the cellular component to be purified is resistant to such cleavage. Efficient degradation of chromosomal D ⁇ A then occurs as the result of the action of one or more exonuclease enzymes acting at the free ends of D ⁇ A generated at the cleavage sites.
  • the restriction enzyme is produced within the cells containing the cellular component to be purified.
  • the restriction enzyme is produced by cells in the medium other than those cells containing the cellular component to be purified.
  • the invention also provides a method of purifying a cellular component substantially free of chromosomal DNA comprising culturing cells producing the cellular component and cells producing a restriction enzyme and then lysing the cells so that the restriction enzyme is allowed access to, and cleaves, the chromosomal DNA of all the cells, whilst the cellular component to be purified is resistant to such cleavage.
  • the cells producing the cellular component also produce the restriction enzyme and when the chromosomal DNA of the cells is exposed to the restriction enzyme it is cleaved, whilst the cellular component to be purified is resistant to such cleavage.
  • the term "cells" or "cell” refers to any prokaryotic or eukaryotic cell(s).
  • Preferred eukaryotic cell(s) include yeasts such as Saccharomyces cereviseae and Staphylococcus spp., mammalian cells such as CHO, BKK and HeLa cells, insect cells such as Sf21 and Sf9 and plant cells.
  • the prokaryotic cell(s) may be any Gram-negative or Gram-positive bacterium, including, but not limited to, Escherichia coli, Salmonella typhimurium, Bacillus spp., Streptomyces spp., and Pseudomonas aeruginosa.
  • cellular component refers to any cellular component except chromosomal DNA.
  • Preferred cellular components include nucleic acid molecules that are resistant to the restriction enzyme such as RNA molecules and DNA molecules such as plasmids, cosmids and artificial chromosomes; proteins, peptides, amino acids, carbohydrates, lipids and glycolipids, whether recombinant or not.
  • resistant means not susceptible to enzymatic cleavage by the restriction enzyme used in the invention. In the case of DNA, resistance is conferred by the absence of the appropriate recognition sequence for the enzyme, or of its chemical modification by methylation or otherwise.
  • the term “recombinant” refers to any cellular component produced in a cell as a result of genetic manipulation that is not naturally produced, or not normally produced in such amounts.
  • the term "substantially free of chromosomal DNA” means that the amount of chromosomal DNA (by weight) is reduced by at least 45%, preferably at least 75% and more preferably at least 95% in the method of the present invention compared to the corresponding method wherein the chromosomal DNA is not exposed to a restriction enzyme. It is particularly preferred that the term "substantially free of chromosomal DNA", especially when an exogenous exonuclease is added or the level of an exonuclease in the cell is higher than normal, means the exceedingly small amount of chromosomal DNA that is permitted in a preparation of a cellular component that is useful for administration to humans or animals.
  • Acceptable exceedingly low levels of chromosomal DNA in samples is less than 1%, preferably less than 0.2% and most preferably less than O.P/o-0.01%) (w/w) of chromosomal DNA to the cellular component.
  • acceptable exceedingly low levels of chromosomal DNA in samples is defined as less than lOng more preferably less than lOpg of chromosomal DNA per dose.
  • the restriction enzyme may be encoded on a chromosome of the cells producing the restriction enzyme or may be encoded on a plasmid within the cells producing the restriction enzyme.
  • the plasmid encoding the restriction enzyme within the cells may or may not be the cellular component to be purified.
  • the restriction enzyme is encoded on the plasmid and the plasmid is the cellular component to be purified.
  • the restriction enzyme is encoded on a first plasmid within the cells and the cellular component to be purified is a second plasmid within the cells.
  • the first and second plasmids may be within the same cells or be in different cells.
  • the first and second plasmids are within the same cells.
  • the expression of the restriction enzyme can be constitutive or regulated expression.
  • regulated or “regulatable” refers to gene expression that is susceptible to control, at the transcriptional or post-transcriptional level, by means of manipulation of environmental conditions.
  • the expression of the restriction enzyme is controlled in a regulated manner.
  • the expression of the restriction enzyme can be controlled in a regulated manner by using an operably-linked inducible promoter or a regulatable operably-linked repressor system.
  • Preferred operably-linked inducible promoters include ⁇ P L , other phage promoters such as ⁇ P R T 3 , T 4 etc., lac, tac, trc, trp and para promoters.
  • Preferred regulatable operably-linked repressor systems include the Lac repressor system and mutations thereof (Betz et al., (1986); Khoury et al, (1991); and Carlos (1978)), the ArgRNN repressor system (Burke et al, (1994)), the E. coli trp repressor (Ingraham et al, (1987)), the E. coli galR repressor (Ingraham et al, (1987)), the E. coli ara C repressor (Ingraham et al, (1987)) and the ⁇ repressor (Hochschild et al., (1986)).
  • Such repressor systems are discussed in European patent application EP-A-0851932.
  • the expression of the restriction enzyme can be controlled in a regulated manner by regulated expression of antisense R ⁇ A.
  • antisense R ⁇ A to regulate gene expression is well known to those skilled in the art and is described in Delihas 1995), Delihas et al, (1997) and Wagner et al, (1994).
  • Figure 1 A schematic method of regulating expression of the restriction enzyme using antisense R ⁇ A is shown in Figure 1.
  • inducible refers to conditional transcriptional activation or derepression.
  • the restriction enzyme may be secreted out of the cells' cytoplasm, for example, secreted into the host cell periplasm or secreted out of the cells into the medium.
  • the chromosomal D ⁇ A is then exposed to the restriction enzyme when the cells are lysed.
  • Suitable secretion signals include the E coli alkaline phosphatase signal peptide (phoA), the pelB sequence and the signal peptide of the OmpA protein of the E. coli outer membrane.
  • phoA E coli alkaline phosphatase signal peptide
  • pelB sequence the signal peptide of the OmpA protein of the E. coli outer membrane.
  • sufficient restriction enzyme activity is present so that when the chromosomal DNA is exposed to the restriction enzyme substantially all of the appropriate recognition sites for the enzyme in the chromosomal DNA are cleaved.
  • substantially all as used herein means that at least 90%, more preferably at least 95% and most preferably at least 99% of the appropriate recognition sites in the
  • Any restriction endonuclease that cuts within, or adjacent to, a specific DNA base recognition sequence and is capable of regulatable expression may be used in the present invention.
  • Such enzymes include Class I enzymes such as Eco K and Eco B (Rosamund, Erium et al. 1979), or Class III enzymes such as Eco PI and Eco PI 5, as well as the more widely-used Class II enzymes including Eco K, Eco B, Eco PI, Eco PI 5, Bam HI, Eco RI, Hind III, Pstl, Kpul and S cl.
  • Class I enzymes such as Eco K and Eco B (Rosamund, Erium et al. 1979)
  • Class III enzymes such as Eco PI and Eco PI 5
  • Bam HI Eco RI
  • Hind III Hind III
  • Pstl Kpul and S cl
  • an enzyme that has a comparatively rare recognition sequence, to reduce the number of such sites that have to be removed during construction of the plasmid.
  • Such enzymes include those with 8 or more bases in their recognition sequences and those recognising sequences containing only A and T, or only C and G. Such sequences are rare in plasmids, but common enough in chromosomal DNA for it to be sufficiently cleaved.
  • enzymes examples include Asc I, Bbv CI, Fse I, Not I, Pac I, Pme I, Rsr II, Sap I, Se* Al, Sfi I, Sgf l, Sgr Al, Sbf l, Srf l, Sse 83871, Swa I, and intron- encoded endonucleases such as I-Cew I and Pl-Psp I.
  • Other enzymes that have a comparatively rare recognition sequence are those which recognise a sequence comprising a dinucleotide sequence which occurs rarely in the chromosomal DNA.
  • the dinucleotide CpG is rare in human chromosomal DNA so that 6 base pair recognition restrictions enzymes such as Xhol, Xmal, Smal, Sal GI, S cII, Pvul, Nurl, Nael, Mlul, Fspl Eagl, Bss HII, Bss KI, Bsp El, Bsm BI, Bsi WI, Agel and Aatll could be used to cleave the chromosomal DNA whilst leaving the cellular component (e.g. plasmid DNA) intact.
  • 6 base pair recognition restrictions enzymes such as Xhol, Xmal, Smal, Sal GI, S cII, Pvul, Nurl, Nael, Mlul, Fspl Eagl, Bss HII, Bss KI, Bsp El, Bsm BI, Bsi WI, Agel and Aatll could be used to cleave the chromosomal DNA whilst
  • the method of the present invention additionally comprises the regulated production of a cognate DNA methylase within the cells that produce the restriction enzyme, such that the DNA cleavage that would otherwise result from the action of the restriction enzyme is reversibly prevented.
  • the expression of the cognate DNA methylase can be controlled in a regulated manner by using an operably-linked inducible promoter, a regulatable operably-linked repressor system or by regulated expression of antisense RNA.
  • Such methods of regulation are discussed above with respect to the expression of the restriction enzyme and are applicable here with respect to the regulation of the methylase.
  • the cognate DNA methylase may be encoded on a chromosome of the cells or on a plasmid within the cells.
  • the plasmid may also encode the restriction enzyme and/or may be the cellular component to be purified.
  • a cognate DNA methylase means a methylase that recognises the same sequence of bases as the restriction enzyme used to cleave the chromosomal DNA.
  • the expression of a cognate DNA methylase is used to prevent cleavage of DNA by said restriction enzyme by methylating one or more bases within the recognition sequence.
  • the regulated expression of the cognate DNA methylase is particularly useful when the restriction enzyme is constitutively expressed as it prevents the chromosomal DNA being cleaved until cleavage is desired. Furthermore, even when the restriction enzyme is being regulatably expressed the regulated expression of the cognate DNA methylase provides a second level of control and therefore reduces the risk of uncontrolled DNA cleavage.
  • Preferred methylases include Eco RI methylase, ApaLl methylase, Sacl methylase, and Seal methylase (Xu et al, (1998)). Other suitable methylases are described in Roberts et al, (1999).
  • the chromosomal DNA previously cleaved in the method of the present invention is exposed to further digestion by an exonuclease.
  • the exonuclease may be added to the cleaved chromosomal DNA exogenously or may be produced by the cells in the medium.
  • the exonuclease may be produced within the cells containing the cellular component to be purified or may be produced within cells in the medium other than those containing the cellular component to be purified.
  • the exonuclease may be encoded in a chromosome of the cells or may be encoded on a plasmid within the cells.
  • the plasmid may also encode the restriction enzyme and/or may be the cellular component to be purified.
  • the exonuclease is present at a higher level than normal in the cells.
  • the term "present at a higher level than normal” refers to the presence of the exonuclease at a level of 10% or more, 50% or more, 100% or more, or even up to over 200-500%) more of the exonuclease than the wild type cell normally produces.
  • the increased levels of exonuclease result from increased expression of the exonuclease.
  • Increased expression of the exonuclease is preferably due to the presence of additional copies of the coding sequence of an exonuclease that naturally occurs in the cell type being used.
  • the increased levels of exonuclease result from the insertion of exogenous regulatory sequences that are then operably-linked to the endogenous exonuclease coding sequence.
  • the chromosomal DNA cleaved by the restriction enzyme is further digested by an exonuclease not normally expressed in the cell type being used, the coding sequence of which has been artificially introduced into the cell.
  • substantially all cells means at least 99%> of the cells present, more preferably at least 99.9%) of the cells present and most preferably at least 99.99%> of the cells present.
  • exonuclease capable of degrading linear DNA can be used in the present invention.
  • Preferred exonucleases include E coli exonucleases that degrade linear DNA fragments, such as exonuclease III, RecBC (exonuclease V), exonuclease VII.
  • Other exonucleases, such as ⁇ exonuclease, may also be used.
  • the present invention further provides a host cell for producing a cellular component substantially free of chromosomal DNA, which produces a restriction enzyme in a regulatable manner for use in the method of the present invention.
  • the present invention also provides a host cell for producing a cellular component substantially free of chromosomal DNA, which produces a restriction enzyme in a constitutive or regulatable manner and produces a cognate DNA methylase in a regulatable manner for use in the method of the present invention.
  • the host cell of the present invention is not capable of DNA repair.
  • the host cell is not capable of repairing DNA cleaved by a restriction enzyme.
  • the host cell is recombinase deficient, so that it does not repair DNA cleaved by restriction enzymes.
  • the host cell is recA ' or uvrA ⁇ .
  • the host cell of the present invention expresses an exonuclease at a higher than normal level for digesting cleaved DNA.
  • the host cell of the present invention contains the cellular component to be purified. If the host cell of the present invention does not contain the cellular component to be purified, it must be used with a cell containing the cellular component to be purified.
  • the present invention further provides a composition comprising the host cell of the present invention which does not contain the cellular component to be purified and a cell containing the cellular component to be purified.
  • the present invention also provides a cellular component purified according to the method of the present invention.
  • the purified cellular component can be nucleic acid molecules that are resistant to the restriction enzyme such as RNA molecules and DNA molecules such as plasmids, cosmids and artificial chromosomes, proteins, peptides, amino acids, carbohydrates, lipids and glycolipids.
  • the purified cellular components is plasmid DNA.
  • the invention is particularly useful in providing preparations that involve administration to humans or animals, but also for other uses such as for biological and biochemical research applications, or any other process where the contribution of chromosomal DNA to the physical properties of cell lysates is deleterious.
  • the invention encompasses a pharmaceutical composition comprising a cellular component that is substantially free of chromosomal DNA obtainable by the method of the present invention, in combination with a pharmaceutically-acceptable carrier.
  • the expression of the restriction enzyme is tightly regulated to prevent inappropriate destruction of chromosomal DNA and killing of the host cells before the cell culture is ready for harvesting. This may be accomplished in a number of ways using a number of features of the present invention.
  • the restriction enzyme may expressed by a very tightly controlled promoter, such as the ⁇ P and ⁇ P R promoters regulated by cl or cl 85 , the Ptrp promoter regulated by the trp repressor, the Plac, Ptac and Ptrp promoters regulated by the lac repressor, and the Para promoter regulated by araC.
  • a very tightly controlled promoter such as the ⁇ P and ⁇ P R promoters regulated by cl or cl 85 , the Ptrp promoter regulated by the trp repressor, the Plac, Ptac and Ptrp promoters regulated by the lac repressor, and the Para promoter regulated by araC.
  • cognate DNA methylase Another level of control of restriction enzyme activity is possible by the use of a cognate DNA methylase wherein expression of cognate DNA methylase activity protects DNA against the cleavage by the restriction enzyme.
  • the cognate DNA methylase may be used with a constitutively expressed restriction enzyme.
  • the cognate DNA methylase is used together with one or both of the above strategies.
  • methylase is induced or derepressed during growth of the culture.
  • endonuclease activity is required to cleave chromosomal DNA
  • expression of the methylase is repressed.
  • DNA synthesised from this point is unmethylated and following two further rounds of replication, double stranded unmethylated DNA is susceptible to cleavage.
  • Figure 2 Shows the amount of chromosomal and plasmid DNA obtained from a first culture over 17 hours.
  • pLV59 encodes both Eco RI and Eco RI methylase with a chloramphenicol resistance marker (O'Connor and Humphreys 1982).
  • the expression of the methylase is temperature- sensitive (28° being permissive), whilst expression of the endonuclease is constitutive.
  • the plasmid is 6.3 kb and itself contains an Eco RI site.
  • pQR261 was constructed from pUC18 by a Pvu II deletion, which removed an approximately 620 bp fragment including the only Eco RI site. It is 2.36 kb and carries an ampicillin resistance marker.
  • the transformed cells were plated onto nutrient agar (Oxoid) containing chloramphenicol 10 mg/ml and ampicillin 500 mg/ml and grown for 2 days at 28 °C.
  • E. coli DH5a containing pLN59 and PQR261 was grown in two tubes each containing 5 mis of nutrient broth (Nutrient Broth No. 2, Oxoid) containing chloramphenicol 10 ⁇ g/ml and ampicillin 500 ⁇ g/ml for 17 hours at 28°C. 4 ml of each was then used to inoculate two flasks containing 300 ml of nutrient broth containing chloramphenicol 10 ⁇ g/ml and ampicillin 500 ⁇ g/ml. The culture was grown at 28°C for 5.5 hours when the OD 600 was 0.423 for culture 1 and 0.38 for culture 2. The flasks were then transferred to 37°C and grown further. 10 ml samples were removed for total DNA and 10 ml for miniprep analysis at various times after transfer to 37°C.
  • Total DNA was prepared as follows. 10 ml cells were centrifuged at 6,000 rpm for 10 min and the cell pellet resuspended in 250 ⁇ l of PI buffer from the Qiagen miniprep kit. 25 ⁇ l of 10%) SDS solution was then added and the tube was vortexed. After 5 minutes, 250 ⁇ l of phenol/chloroform/isoamyl alcohol 25:24:1 (Sigma P-3803) was added along with 250 ⁇ l of H 2 O and the mixture was vortexed thoroughly. The tubes were centrifuged at 14,000 rpm for 10 minutes and the upper layer was removed to a clean tube. 50 ⁇ l of 5M NaCl was added plus 1 ml of absolute ethanol.
  • the tubes were mixed by vortexing and left to precipitate for 30 minutes at room temperature.
  • the tubes were centrifuged at 14,000 rpm for 5 minutes, the DNA pellets washed with 500 ⁇ l ethanol and dried.
  • the DNA was resuspended in 300 ⁇ l of 10 mM Tris.HCl buffer pH 7.5, 1 mM ⁇ DTA.
  • Samples of the total DNA from the above culture were electrophoresed on 1% agarose gels with Tris Borate electrophoresis buffer containing 0.5 ⁇ g/ml ethidium bromide.
  • the gels were visualised using uv light and the image captured using UVITec photodocumentation camera system and UVIDoc and UNIBand quantitation V.97 software (UVITec Ltd).
  • the amount of D ⁇ A sample run on the gel was adjusted for the OD 600 value of the culture at each time point. Equal OD 600 equivalents were run on the gels.

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Abstract

L'invention concerne un procédé de purification d'un composant cellulaire, sensiblement dépourvu d'ADN chromosomique. Ce procédé consiste à cultiver, dans un milieu, des cellules produisant le composant cellulaire, puis à exposer l'ADN chromosomique des cellules à une enzyme de restriction de manière que cet ADN soit clivé, le composant cellulaire à purifier étant résistant à un tel clivage.
PCT/GB2000/004006 1999-10-21 2000-10-19 Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique WO2001029209A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
KR1020027004874A KR20020064884A (ko) 1999-10-21 2000-10-19 실질적으로 염색체 디엔에이가 없는 세포 구성성분의 제조
JP2001532192A JP2003512054A (ja) 1999-10-21 2000-10-19 染色体ｄｎａを実質的に含まない細胞成分の調製
EP00971521A EP1222260A1 (fr) 1999-10-21 2000-10-19 Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique
AU10361/01A AU1036101A (en) 1999-10-21 2000-10-19 Preparation of cellular components that are substantially free of chromosomal dna
CA002386401A CA2386401A1 (fr) 1999-10-21 2000-10-19 Preparation de composants cellulaires sensiblement depourvus d'adn chromosomique
NO20021782A NO20021782L (no) 1999-10-21 2002-04-16 Fremstilling av cellul¶re komponenter som er vesentlig frie for kromosomalt DNA
HK02105760.5A HK1045539A1 (zh) 1999-10-21 2002-08-06 基本上無染色體dna的細胞成分的製備方法

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GBGB9924973.2A GB9924973D0 (en) 1999-10-21 1999-10-21 Preparation of cellular components that are substantially free of chromosomal DNA
GB9924973.2 1999-10-21

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8748168B2 (en) 2004-08-16 2014-06-10 Nature Technology Corp. Strains of E. coli for plasmid DNA production

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992013963A1 (fr) * 1991-01-30 1992-08-20 Hyman Edward D Procede de preparation d'adn circulaire ferme
US5470706A (en) * 1988-07-19 1995-11-28 Nederlandse Organisatie Voor Toegepastwetenschappelijk Onderzoek Tno Process for the rescue of DNA and for detecting mutations in marker genes
WO1997029190A1 (fr) * 1996-02-12 1997-08-14 Cobra Therapeutics Limited Procede pour la production et la purification d'adn plasmide
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CA2386401A1 (fr) 2001-04-26
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AU1036101A (en) 2001-04-30
EP1222260A1 (fr) 2002-07-17
NO20021782D0 (no) 2002-04-16
NO20021782L (no) 2002-06-13
JP2003512054A (ja) 2003-04-02
KR20020064884A (ko) 2002-08-10

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