PREPARATION OF CELLULAR COMPONENTS THAT ARE SUBSTANTIALLY FREE OF CHROMOSOMAL DNA

Abstract: The invention relates to a method of purifying a cellular component substantially free of chromosomal DNA. The method comprises culturing cells producing the cellular component in a medium. The chromosomal DNA of the cells is then exposed to a restriction enzyme such that it is cleaved. The cellular component to be purified is resistant to such cleavage.

Description: PREPARATION OF CELLULAR COMPONENTS THAT ARE SUBSTANTIALLY FREE OF CHROMOSOMAL DNA 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'. In the case of many restriction enzymes, 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). However, on a commercial scale, 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). In this case, 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 DΝA molecules. The purpose of this so-called "maxicell" technique is to identify plasmid-encoded, radiolabelled proteins in cell extracts. It does not teach any method of improving plasmid DΝA purification, and does not use restriction enzymes to cleave chromosomal DΝA. Furthermore, the use of ultraviolet irradiation is difficult, as all the cells need to be exposed to sufficient ultraviolet irradiation to cause DΝA cleavage. The use of ultraviolet irradiation is therefore not practicable when dealing with a large culture of cells. Accordingly, there is a need in the art for a simple and efficient method of removing chromosomal DΝA from cellular components. The present invention provides a method of purifying a cellular component substantially free of chromosomal DΝA comprising culturing cells producing the cellular component in a medium and then exposing the chromosomal DΝA 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 DΝA then occurs as the result of the action of one or more exonuclease enzymes acting at the free ends of DΝA generated at the cleavage sites. In a preferred embodiment, the restriction enzyme is produced within the cells containing the cellular component to be purified. Alternatively, the restriction enzyme is produced by cells in the medium other than those cells containing the cellular component to be purified. In one embodiment, 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. In an alternative embodiment, 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. As used herein, 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. As used herein, the term "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. As used herein, the term "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. As used herein, 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. As used herein, 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. Alternatively, 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. In the method of the present invention, 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. In a preferred embodiment, the restriction enzyme is encoded on the plasmid and the plasmid is the cellular component to be purified. In an alternatively preferred embodiment, 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. Preferably, the first and second plasmids are within the same cells. The expression of the restriction enzyme can be constitutive or regulated expression. As used herein, the term "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. In preferred embodiments of the above-described inventive methods, 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 λP L , other phage promoters such as λP 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 λ repressor (Hochschild et al., (1986)). Such repressor systems are discussed in European patent application EP-A-0851932. Alternatively, the expression of the restriction enzyme can be controlled in a regulated manner by regulated expression of antisense RΝA. The use of antisense RΝA 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). A schematic method of regulating expression of the restriction enzyme using antisense RΝA is shown in Figure 1. As used herein, the term "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 DΝA 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. Preferably 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. The term "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 chromosomal DNA for the enzyme are cleaved. 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, Endlich 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. However, when the cellular product is DNA, such as a plasmid, then the DNA e.g. plasmid, itself and any insert it carries must be free of the recognition sequence of the restriction enzyme being used. It may be preferable, in these circumstances, to use 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. Examples of such enzymes are 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. For example, 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. In a further preferred embodiment 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. When the methylase is encoded on a plasmid, the plasmid may also encode the restriction enzyme and/or may be the cellular component to be purified. The term "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). Preferably 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. When the exonuclease is encoded on a plasmid, the plasmid may also encode the restriction enzyme and/or may be the cellular component to be purified. Preferably 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. Preferably, 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. Alternatively, the increased levels of exonuclease result from the insertion of exogenous regulatory sequences that are then operably-linked to the endogenous exonuclease coding sequence. In a further alternative embodiment, 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. When the exonuclease is produced within the cells in the medium other than those containing the cellular component to be purified, lysis of the cells in the medium allows access of the exonuclease to the DNA of substantially all cells present. As used herein, the term "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. Any 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 λ 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. Preferably, the host cell of the present invention is not capable of DNA repair. Preferably, the host cell is not capable of repairing DNA cleaved by a restriction enzyme. More preferably, the host cell is recombinase deficient, so that it does not repair DNA cleaved by restriction enzymes. Preferably, the host cell is recA ' or uvrA ~ . Preferably, the host cell of the present invention expresses an exonuclease at a higher than normal level for digesting cleaved DNA. Preferably, 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. Preferably 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. In the present invention, it is important that 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. 1. The restriction enzyme may expressed by a very tightly controlled promoter, such as the λP and λP 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. 2. The regulation by such promoters may be further tightened by the use of an antisense strategy, as follows (Figure 1). An antisense transcript produced at a low level from a weak constitutive promoter binds specifically to the ribosome binding site and 5' end of the restriction enzyme gene. All transcription of restriction enzyme is blocked, since any opening of the DNA causes high affinity binding of the antisense RNA to restriction enzyme mRNA and the positive strand of the coding DNA strand. Upon induction or derepression of the promoter, however, the low levels of antisense transcript are titrated out and active expression of the restriction enzyme may proceed. 3. 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. Preferably the cognate DNA methylase is used together with one or both of the above strategies. Expression of the methylase is induced or derepressed during growth of the culture. When 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. Further features and advantages of the invention will become more fully evident in the following description and drawings of the embodiments, and from the claims. The drawings are briefly described. Figure 1 Illustration of the use of an antisense transcript for tight control of restriction enzyme expression. Figure 2 Shows the amount of chromosomal and plasmid DNA obtained from a first culture over 17 hours. Figure 3 Shows the amount of chromosomal and plasmid DNA obtained from a second culture over 17 hours. EXAMPLE Method E. coli DH5α were co-transformed with plasmids pLV59 and pQR261. 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 μg/ml and ampicillin 500 μg/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 μg/ml and ampicillin 500 μg/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 μl of PI buffer from the Qiagen miniprep kit. 25 μl of 10%) SDS solution was then added and the tube was vortexed. After 5 minutes, 250 μl of phenol/chloroform/isoamyl alcohol 25:24:1 (Sigma P-3803) was added along with 250 μl 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 μl 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 μl ethanol and dried. The DNA was resuspended in 300 μl of 10 mM Tris.HCl buffer pH 7.5, 1 mM ΕDTA. Samples of the total DNA from the above culture were electrophoresed on 1% agarose gels with Tris Borate electrophoresis buffer containing 0.5 μg/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 DΝA 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. The results are shown in Figures 2 and 3, wherein the values for the amount of DΝA in the chromosomal DΝA band and the plasmid band are amounts of pixels. The results show that the amount of chromosomal DNA in the samples is reduced over time and that the amount of plasmid DNA is maintained. It will be appreciated that increasing the level of exonuclease present in accordance with the present invention can further reduce the amount of chromosomal DNA present. REFERENCES Betz et al, (1986) Gene 42:283-292. Bi nboim, H. and J. Doly (1979). "A rapid alkaline extraction procedure for screening recombinant plasmid DNA." Nucleic Acids Research 7: 1513-1523. Burke et al, (1994) Mol. Microbiol. 13: 609-618. Carlos (1978) Nature 274: 762-765. Delihas (1995) Mol. Microbiol. 15: 411-414. Delihas et al, (1997) Nature Biotechnology 15: 751-753. Hill, R. T., N. Illing, et al. (1989). "Development of pLR591, a Streptomyces - Escherichia coli positive selection shuttle vector." FEMS Microbiology Letters 57: 223-226. Hochscild et al, (1986) CeU 47: 807-816. Ingraham et al, (1987) Amer. Soc. Micro. 81: 1453-1481 and 1503-1512 . Khoury et al, (1991) J. Mol. Bio 219: 623-634. Kuhn, I., F. H. Stephenson, et al. (1986). "Positive-selection vectors utilizing lethality of the Eco RI endonuclease." Gene 44: 253-263. Lewin, B. (1983). Genes. New York, John Wiley & Sons. O'Connor, C. and G. Humphreys (1982). "Expression of the Eco RI restriction- modification system and the construction of positive-selection cloning vectors." Gene 20: 219-229. Roberts et al. NAR 27: 312-313. Rosamund, J., B. Endlich, et al. (1979). "Electron microscopic studies of the mechanism of action of the restriction endonuclease of Escherichia coli B." Journal of Molecular Biology 129: 619-635. Sambrook, J., E. F. Fritsch, et al. (1989). Molecular cloning: a laboratorv manual. New York, Cold Spring Harbor Laboratory Press. Sancar, A., A. M. Hack, et al. (1979). "Simple method for identification of plasmid- encoded proteins." Journal of Bacteriology 137(1): 692-693. Wagner et al, Ann. Rev. Microbiol. 48: 713-742. Xu et al. Mol. Gen. Genet. 260: 226-231.

Claims: Claims 1. A method of purifying a cellular component substantially free of chromosomal DNA comprising culturing cells producing the cellular component in a medium and then exposing the chromosomal DNA of the cells to a restriction enzyme such that it is cleaved, whilst the cellular component to be purified is resistant to such cleavage. 2. The method of claim 1 wherein the restriction enzyme used to cleave the chromosomal DNA is produced within the cells containing the cellular component to be purified. 3. The method of claim 1 or claim 2 wherein the restriction enzyme is encoded on the chromosome of the cells containing the cellular component to be purified. 4. The method of claim 1 or claim 2 wherein the restriction enzyme is encoded on a plasmid within the cells containing the cellular component to be purified. 5. The method of claim 4 wherein the plasmid encoding the restriction enzyme is the cellular component to be purified. 6. The method of claim 1 wherein the restriction enzyme used to cleave the chromosomal DNA is produced by cells in the medium other than those cells containing the cellular components to be purified 7. The method of claim 6 wherein the cells producing the cellular component and cells producing a restriction enzyme are cultured and then lysed 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. 8. The method of claim 6 or claim 7 wherein the restriction enzyme is encoded on the chromosome of the cells producing said restriction enzyme. 9. The method of claim 6 or claim 7 wherein the restriction enzyme is encoded on a plasmid within the cells producing said restriction enzyme. 10. The method of any of the preceding claims wherein the expression of the restriction enzyme is regulated by an operably-linked inducible promoter. 11. The method of claim 10 wherein the operably-linked promoter is λP L , λP R , T 3 , T 4 , lac, tac, trc, trp or para. 12. The method of any of claims 1 to 9 wherein the expression of the restriction enzyme is regulated by means of a regulatable operably-linked repressor system. 13. The method of claim 14 wherein the regulatable operably-linked repressor system is the Lac repressor system, the ArgRNV repressor system, the E. coli trp repressor, the E. coli galR repressor, the E. coli ara C repressor or the λ repressor. 14. The method of claim 14 wherein expression of the restriction enzyme is reversibly repressed by regulated expression of antisense RNA. 15. The method of any of the preceding claims wherein the restriction enzyme is any of the following: Eco K, Eco B, Eco PI, Eco PI 5, Bam HI, Eco RI, Hind III, Psrl, Kpul, Sacl, Asc I, Bbv CI, Fse I, Not I, Pac I, Pme I, R^r II, Sap I, Sex Al, Sfi I, Sgfl, Sgr Al, Sbfl, Srfl, Sse 83871, Swa I, , Xhol, Xmal, Smal, Sal GI, SαcII, Pvul, Nurl, Nael, MM, Fspl Eagl, Bss HII, Bss KI, Bsp ΕI, Bsm BI, Bsi WI, Agel, Aatll and intron- encoded endonucleases such as l-Ceu I and Vl-Psp I. 16. The method of any one of the preceding claims wherein the cellular component to be purified is a nucleic acid molecule that is resistant to the restriction enzyme, protein, peptide, amino acid, carbohydrate, lipid or glycolipid. 17. The method of claim 16 wherein the nucleic acid molecule is a plasmid, cosmid or a artificial chromosome. 18. The method of any of the preceding claims comprising the regulated expression 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. 19. The method of claim 18 wherein the methylase is encoded on the chromosome of the cell. 20. The method of claim 18 wherein the methylase is encoded on a plasmid. 21. The method of any of claims 18 to 20 wherein the methylase is Eco RI methylase. 22. The method of any of the preceding claims comprising exposure of the chromosomal DNA previously cleaved by the restriction enzyme to further digestion by an exonuclease. 23. The method of claim 22 wherein the exonuclease is present at a higher level than normal in the cells. 24. Cellular components purified according to the method of any one of the preceding claims. 25. A host cell for producing a cellular component substantially free of chromosomal DNA, which produces a restriction enzyme in a regulatable manner suitable for use in the method of any one of claims 1 to 23. 26. 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 any one of claims 18 to 23. 27. The host cell of claim 25 or claim 26 that is recombinase deficient. 28. The host cell strain of claim 27 that is recA. 29. The host cell of any one of claims 24 to 28 expressing an exonuclease suitable for use in the method of any one of claims 22 to 23. 30. A composition comprising the host cell of any one of claims 24 to 29 which does not contain the cellular component to be purified and a host cell containing the cellular component to be purified. 31. A pharmaceutical composition comprising a cellular component that is substantially free of chromosomal DNA obtainable by the method of any one of claims 1 to 23 in pharmaceutically acceptable carrier.

Inventors: HANAK JULIAN ALEXIS JOHN, WARD JOHN MAXIM, HANAK, JULIAN, ALEXIS, JOHN, WARD, JOHN, MAXIM
Applicants: COBRA THERAPEUTICS LTD, HANAK JULIAN ALEXIS JOHN, WARD JOHN MAXIM, HANAK, JULIAN, ALEXIS, JOHN, WARD, JOHN, MAXIM
Application year: 2000
Main classification: C12N15
IPC codes: A61K31, A61K35, A61P43, C12N1, C12N5, C12N15