WO2005113808A1 - Procede de fabrication de plasmagène par conversion de plasmagène circulaire ouvert en plasmagène surfondu - Google Patents

Procede de fabrication de plasmagène par conversion de plasmagène circulaire ouvert en plasmagène surfondu Download PDF

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WO2005113808A1
WO2005113808A1 PCT/US2004/014946 US2004014946W WO2005113808A1 WO 2005113808 A1 WO2005113808 A1 WO 2005113808A1 US 2004014946 W US2004014946 W US 2004014946W WO 2005113808 A1 WO2005113808 A1 WO 2005113808A1
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plasmid
dna
open circular
exonuclease
supercoiled
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PCT/US2004/014946
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English (en)
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Edward D. Hyman
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Hyman Edward D
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Priority to PCT/US2004/014946 priority Critical patent/WO2005113808A1/fr
Priority to US10/947,360 priority patent/US20050255563A1/en
Priority to US11/231,636 priority patent/US7510856B2/en
Publication of WO2005113808A1 publication Critical patent/WO2005113808A1/fr

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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
    • 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

  • Plasmids are double stranded, circular, extrachromosomal DNA molecules
  • Plasmids are defined as such herein. Plasmids are contained inside host cells. One common host cell is Escherichia coli (E. coli). Many other types of cells are known to carry plasmids. This includes other bacteria, yeast, and higher eukaryotic cells. Plasmids may be artificial (i.e., manmade), such as cloning vectors carrying foreign DNA inserts. Plasmids may also occur naturally, such as in mitochondria and chloroplasts. Since the invention of cloning circa 1975, the preparation of plasmid has been a routine task in molecular biology. Plasmid preparation has become a highly crowded art. The crowded nature of the art is a reflection of the widespread importance of the procedure in molecular biology.
  • plasmid preparation Numerous articles and patents have been published in the past 25 years describing novel methods for preparing plasmid.
  • the problem of plasmid preparation has attracted enormous commercial interest.
  • Companies sell kits for plasmid preparation (Amersham, Qbiogene, Clonetech, Promega, Biorad, Qiagen, Sigma); proprietary resins for purifying plasmid (Qiagen, Amersham, Puresyn, Macherey-Nagel); and automated instruments for preparing plasmid (Qiagen, MacConnell, Autogen).
  • the final plasmid preparation is usually a mixture of two main forms of plasmid: open circular and supercoiled.
  • the plasmid In the supercoiled form, the plasmid has a covalently closed circular form, and the plasmid is negatively supercoiled in the host cell by the action of host enzymes.
  • the open circular form one strand of the DNA duplex is broken at one or more places. The single strand break(s) in an open circular plasmid results in a relaxed topology. Open circular plasmid in a plasmid preparation can result from several causes.
  • Open circular plasmid may exist in the host cells immediately prior to lysis. Some supercoiled plasmid in the host cells may unintentionally be converted to open circular plasmid in the preparation of a cleared lysate, due to the fragile nature of supercoiled plasmid. Additional plasmid purification procedures, such as organic extraction, precipitation, ultrafiltration, and chromatography, may unintentionally convert some supercoiled plasmid from the cleared lysate to open circular plasmid, due to the fragile nature of supercoiled plasmid.
  • open circular plasmid refers to the open circular plasmid which is commonly present in plasmid preparations after purifying plasmid contained in host cells, and does not refer to open circular plasmid which is purposefully synthesized by an in vitro method.
  • Such purposeful in vitro synthetic methods may be enzymatic or nonenzymatic reactions.
  • Non-limiting examples of purposeful in vitro synthesis of open circular plasmid include purposeful in vitro plasmid replication forming open circular daughter plasmids, open circular plasmid purposefully synthesized from single stranded circular DNA by in vitro enzymatic reactions or synthetic primer annealing, and open circular plasmid produced by purposeful conversion of supercoiled plasmid to open circular plasmid such as purposeful damage with free radicals.
  • the active plasmid form is supercoiled.
  • Open circular plasmid is often either inactive or poorly active. Plasmid for gene transfer (e.g.
  • Prior art methods for purifying supercoiled plasmid from open circular plasmid involve separation and removal of open circular plasmid from supercoiled plasmid, or selective degradation of the open circular plasmid.
  • the open circular plasmid is separated and removed.
  • the enzymatic prior art methods open circular plasmid is selectively degraded by exonuclease.
  • One disadvantage of prior art approaches is that the final yield of supercoiled plasmid is reduced because open circular plasmid is removed or degraded.
  • the invention overcomes the inherent disadvantage of prior art methods by using a fundamentally different operating principle, by converting open circular plasmid to supercoiled plasmid.
  • This invention provides an improved method for plasmid preparation.
  • An objective of the invention is to provide a method for preparing supercoiled plasmid, by converting open circular plasmid into supercoiled plasmid enzymatically, thereby achieving a final plasmid preparation which has an increased proportion of supercoiled plasmid.
  • a method for preparing plasmid from host cells which contain the plasmid comprising: (a) providing a plasmid solution comprised of unligatable open circular plasmid; (b) reacting the unligatable open circular plasmid with one or more enzymes and appropriate nucleotide cof actors, such that at least some unligatable open circular plasmid is converted to 3'-hydroxyl, 5'- phosphate nicked plasmid; (c) reacting the 3'-hydroxyl, 5 '-phosphate nicked plasmid with a DNA ligase and DNA ligase nucleotide cofactor, such that at least some 3'- hydroxyl, 5 '-phosphate nicked plasmid is converted to relaxed covalently closed circular plasmid; and (d) reacting the relaxed covalently closed circular plasmid with a DNA gyrase and DNA gyrase nucleotide cofactor, such that at least
  • DNA gyrase is replaced by reverse DNA gyrase or reaction (d) is not performed.
  • Incubations may also include salt, buffer, and nucleotide cofactor appropriate for the enzyme.
  • Reaction conditions such as concentration of the aforementioned chemicals, temperature, and time may be adjusted to provide suitable conversion kinetics and yield.
  • reactions (b), (c), and (d) are performed in a single reaction using an enzyme mixture comprising a DNA polymerase, DNA ligase, and DNA gyrase.
  • the mixture further comprises a 3' deblocking enzyme.
  • the mixture further comprises a kinase enzyme and a high energy phosphate donor, which converts the nucleotide by-product of DNA gyrase nucleotide cofactor back to nucleotide cofactor.
  • the enzyme mixture further comprises one or more exonucleases, which degrades linear chromosomal DNA.
  • kits and compositions comprising one or more of the aforementioned enzymes and optional reaction components (e.g. salt, buffer, nucleotide cofactor).
  • enzymes in one or more containers may be packaged for single or multiple reactions.
  • Instructions for practicing a method of the invention are another optional component of the kit. Instructions may be a printed sheet included in the kit or a label applied to the outside of the kit.
  • open circular plasmid is enzymatically converted to supercoiled plasmid. This is accomplished by incubating the open circular plasmid with enzymes, either sequentially or preferably simultaneously with an enzyme mixture. The result of this enzymatic incubation is a plasmid preparation with a higher percentage of supercoiled plasmid and a lower percentage of open circular plasmid.
  • the invention operates in a fundamentally different manner from the prior art.
  • the enzymatic conversion reactions (conversion reactions) of the invention are preferably performed after obtaining a cleared lysate of host cells containing the plasmid.
  • a "cleared lysate” is a well known term in the art and refers to an aqueous solution containing plasmid, and usually RNA, usually soluble proteins, and usually residual amounts of chromosomal DNA, which is obtained after lysis of host cells and the separation of the cell debris, usually by filtration or centrifugation. Any method for preparing a cleared lysate may be potentially useful.
  • Plasmid in the cleared lysate is usually a mixture of supercoiled and open circular plasmid.
  • the host cells containing plasmid are preferably bacteria, preferably Escherichia coli.
  • Two methods are commonly used in the art for producing a cleared lysate from bacteria. Both methods comprise lysing the host cells, precipitating chromosomal DNA, and removing the precipitated chromosomal DNA and cell debris.
  • alkaline lysis method Bornboim, Nucl. Acids Res. 7:1513-1523, 1975
  • host cells are lysed using an alkaline solution.
  • Chromosomal DNA is precipitated by neutralizing the lysed cell solution.
  • the precipitated chromosomal DNA and cell debris is removed by filtration or centrifugation.
  • the boiling method Holmes, Anal. Biochem.
  • host cells are lysed using lysozyme.
  • Chromosomal DNA is precipitated by heating the lysed cell solution; the precipitated chromosomal DNA and cell debris is removed by centrifugation.
  • Other non-limiting methods of potential use for preparing a cleared lysate may include mechanical disruption methods (U.S. Patent 6,455,287).
  • a preferred method for preparing a cleared lysate is the alkaline lysis method. After preparing the cleared lysate, the plasmid in the cleared lysate is optionally further purified from other host cell components in any desired manner prior to the conversion reactions.
  • Further purification can be accomplished by many methods, such as organic solvent extraction, precipitation, RNA digestion by a ribonuclease, chromatography, electrophoresis, ultrafiltration (e.g. tangential flow ultrafiltration), or combinations thereof.
  • the further purification procedure(s) do not separate open circular plasmid from supercoiled plasmid or degrade open circular plasmid.
  • Further purification may be advantageous. Further purification may result in plasmid in a buffer which is more suitable for the conversion reactions. Further purification may allow more efficient and reliable conversion reactions by removing contaminants (such as protein and RNA) which might inhibit the conversion reactions.
  • the resulting plasmid solution comprises open circular plasmid, and usually supercoiled plasmid (i.e., usually a mixture of open circular and supercoiled plasmids).
  • the enzymatic conversion reactions are preferably performed on open circular plasmid in the plasmid solution.
  • One embodiment of the invention preferably comprises at least three enzymatic conversion reactions, which convert unligatable open circular plasmid to supercoiled plasmid. They may be performed sequentially or simultaneously.
  • First Enzymatic Reaction Conversion of unligatable open circular plasmid to 3'- hydroxyl, 5 '-phosphate nicked plasmid.
  • first reaction unligatable open circular plasmid in a plasmid solution is converted in vitro to 3'-hydroxyl, 5 '-phosphate nicked plasmid (ligatable form).
  • Preferred Mode In a preferred conversion method, the unligatable open circular plasmid is converted to 3'-hydroxyl, 5 '-phosphate nicked plasmid by incubation with a DNA polymerase in the presence of deoxyribonucleoside triphosphate substrates (dNTPs).
  • dNTPs deoxyribonucleoside triphosphate substrates
  • a preferred polymerase is DNA polymerase I, which preferably has both 3 '-5' and 5 '-3' exonuclease activities.
  • the 5 '-3' exonuclease activity of DNA polymerase I may advantageously convert some 5' termini that lack a 5 '-phosphate to a 5 '-phosphate terminus. This activity is also known as nick translation.
  • the 3 '-5' exonuclease activity of DNA polymerase I may advantageously convert some 3' termini that lack a 3'- hydroxyl to a 3'-hydroxyl.
  • DNA polymerase I in the presence of dNTPs, converts most of the unligatable open circular plasmid to 3'- hydroxyl, 5 '-phosphate nicked plasmid.
  • Example 1 demonstrates non-limiting embodiments of the preferred mode.
  • Other DNA polymerases may be used.
  • a 3' deblocking enzyme may optionally be used to assist in the first reaction.
  • Some unligatable open circular plasmid may have a blocking group at the 3' terminus.
  • the blocking group may inhibit (completely or partially) the ability of DNA polymerase to extend the 3' terminus.
  • a 3' deblocking enzyme can remove the 3' blocking group and produce a 3'-hydroxyl terminus. The resulting 3'-hydroxyl terminus may then be extended by DNA polymerase.
  • Incubations with 3' deblocking enzyme and DNA polymerase are preferably performed simultaneously, but could also be performed sequentially in the order 3' deblocking enzyme followed by DNA polymerase.
  • Non- limiting examples of 3' deblocking enzymes include 3 '-5 exonucleases, endonucleases (e.g. AP endonucleases), 3'-phosphodiesterase, and phosphatases, and are discussed below.
  • a preferred 3' deblocking enzyme is a 3 '-5' exonuclease, such as preferably exonuclease IE.
  • Exonuclease El converts 3 '-blocked open circular plasmid to 3'- hydroxyl gapped plasmid.
  • Exonuclease IE has four activities, all of which may serve a 3' deblocking function: 3'-5' exonuclease activity, 3'-phosphatase activity, apurinic / apyrimidinic (AP) endonuclease activity and 3'-phosphodiesterase.
  • exonuclease El When coincubated with DNA polymerase, the ratio of exonuclease El and DNA polymerase activities should be balanced appropriately to avoid significant exonuclease degradation of open circular plasmid.
  • Exonuclease IE from any source may be useful. Exonuclease El is likely found in many organisms. A preferred source of exonuclease El is E. coli. Other 3 '-5 exonucleases may also serve as a 3' deblocking enzyme, preferably having low processivity. Another useful 3' deblocking enzyme is an endonuclease, such as preferably AP endonuclease.
  • AP endonuclease converts AP sites in open circular plasmid to 3' hydroxyl gapped plasmid.
  • AP endonucleases are found in many organisms. AP endonuclease from any source may be used. A preferred AP endonuclease is endonuclease IV. A preferred source of endonuclease IV is E. coli. Another useful AP endonuclease may be APE1 (Ranalli, J. Biol. Chem. 277:41715-41724, 2002; Izumi et al. Carcinogenesis 21:1329-1334, 2000).
  • AP endonucleases or other types of endonucleases may also serve as 3' deblocking enzymes.
  • Another useful 3' deblocking enzyme is phosphatase, such as preferably 3'- phosphatase.
  • 3 '-Phosphatase efficiently dephosphorylates a 3 '-phosphate blocking group to 3 '-hydroxyl terminus.
  • Another useful 3 '-phosphatase 3 '-deblocking enzyme is polynucleotide kinase - 3 '-phosphatase (PNKP).
  • the polynucleotide kinase activity of PNKP is able to convert 5 '-hydroxyl termini to 5'-phosphate termini.
  • Other phosphatases may also be useful.
  • Other 3' deblocking enzymes can be used provided that they convert the blocked 3' terminus of open circular plasmid to a 3' hydroxyl terminus. More than one 3' deblocking enzyme may be used during the first reaction. A 3' deblocking enzyme may be especially advantageous when used with a DNA polymerase which lacks 3 '-5 exonuclease activity.
  • a 3' deblocking enzyme may be used with a DNA polymerase which has 3 '-5' exonuclease activity, possibly enhancing repair efficiency.
  • Example 2 demonstrates non-limiting embodiments using 3' deblocking enzymes.
  • Example 2 demonstrates that the 3' deblocking enzymes enhance the conversion efficiency.
  • a 5' deblocking enzyme may optionally be used to assist in the first reaction.
  • the 5' deblocking enzyme converts a blocked 5'-terminus to a 5'-phosphate terminus.
  • the 5' deblocking enzyme may be able to remove 5' blocking groups which DNA polymerase is unable to remove.
  • a preferred 5' deblocking enzyme is flap endonuclease, an enzyme which is homologous to the 5 '-3' exonuclease of DNA polymerase I.
  • DNA polymerase and flap endonuclease are employed for repair of some single strand breaks (Lieber, Bioessays 19:233-240, 1997; Kim, J. Biol. Chem. 273:8842-8848, 1998; Shu, Trends Biochem Sci. 23:171-173, 1998).
  • Incubation with 5' deblocking enzyme and DNA polymerase are preferably performed simultaneously, but could potentially also be performed sequentially in the order: 5' deblocking enzyme followed by DNA polymerase.
  • Non- limiting examples of 5' deblocking enzymes of potential use may include 5 '-3' exonucleases, AP lyases, flap endonucleases or flap exonucleases (such as FEN1 or T5 exonuclease), and DNA deoxyribophosphodiesterases. These enzymes are well characterized in the art of DNA repair (Friedberg et al. DNA Repair and Mutagenesis, ASM Press, 1995). Other 5' deblocking enzymes may potentially be used provided that they convert a blocked 5' terminus of open circular plasmid to a 5' phosphate terminus. A 5' deblocking enzyme may advantageously reduce unintentional strand displacement side reactions of DNA polymerase or remove the displaced strand.
  • a 5' deblocking enzyme may possibly also selectively digest some linear chromosomal DNA. More than one 5' deblocking enzyme may be used in the first reaction. A 5' deblocking enzyme may be especially advantageous when used with a DNA polymerase which lacks 5' terminus repair activity, such as 5 '-3' exonuclease activity. A 5' deblocking enzyme may be used with a DNA polymerase which has 5' terminus repair activity, possibly enhancing repair efficiency.
  • the first reaction may optionally employ both 5' and 3' deblocking enzymes, simultaneously or in any order, but preferably simultaneously with DNA polymerase incubation.
  • the DNA polymerase has both 3' and 5' terminus repair activities (e.g. some DNA polymerase I enzymes).
  • a 3' deblocking or 5' deblocking enzyme or both may optionally be added to possibly enhance repair efficiency.
  • the DNA polymerase lacks 3' terminus repair activity and has 5' terminus repair activity (e.g. Taq DNA polymerase, some eukaryotic DNA polymerases)
  • the first reaction is performed using DNA polymerase and a 3' deblocking enzyme.
  • a 5' deblocking enzyme may optionally be added to possibly enhance repair efficiency.
  • the 5' deblocking enzyme flap endonuclease may be especially advantageous in assisting the inherent 5' terminus lyase repair activity of some eukaryotic DNA polymerases, such as DNA polymerase beta (Wilson, Mut. Res; 407:203-215, 1998; Wilson, Mut. Res.
  • the first reaction may be performed using AP endonuclease, DNA polymerase beta, and optionally flap endonuclease (Wilson, C.S.H. Symp. Quant. Biol., LXV: 143-155, 2000).
  • the DNA polymerase has 3' terminus repair activity and lacks 5' terminus repair activity (e.g. some phage DNA polymerases)
  • the first reaction is performed using DNA polymerase and 5' deblocking enzyme.
  • a 3' deblocking enzyme may optionally be added to possibly enhance repair efficiency.
  • the DNA polymerase lacks both 3' and 5' terminus repair activities (e.g. mutant DNA polymerases), then preferably the first reaction is performed using DNA polymerase, 3' deblocking enzyme, and 5' deblocking enzyme.
  • DNA polymerase e.g. DNA polymerase
  • 3' deblocking enzyme e.g. 3' deblocking enzyme
  • 5' deblocking enzyme e.g. 3' deblocking enzyme
  • a person skilled in the art may optionally select appropriate 3' deblocking and/or 5' deblocking enzymes based on the known enzyme activities of the DNA polymerase, the known in vivo system of the DNA polymerase for single strand break repair, and the desired conversion efficiency of open circular to supercoiled plasmid. It will be appreciated that some DNA polymerases may be advantageous if the DNA polymerase functions in vivo in DNA repair.
  • At least one repair activity is provided in the first reaction for both the 3' terminus and the 5' terminus of open circular plasmid, using either the repair activity from the DNA polymerase or a deblocking enzyme or both.
  • the first reaction most or nearly all unligatable open circular plasmid can be converted to 3'-hydroxyl, 5'-phosphate nicked plasmid.
  • Alternate Mode In an alternate conversion method, the unligatable open circular plasmid is incubated with polynucleotide kinase and 3 '-phosphatase in the presence of nucleotide cofactor, preferably using the enzyme PNKP.
  • PNKP converts unligatable open circular plasmid which is 3 '-phosphate, 5 '-hydroxyl nicked plasmid to 3 '-hydroxyl, 5 '-phosphate nicked plasmid.
  • the incubations with 3 '-phosphatase and polynucleotide kinase are preferably performed simultaneously using PNKP, but could also be performed sequentially in any order.
  • Example 6 demonstrates a non-limiting embodiment of the alternate mode. Using the alternate mode of the first reaction, at least some of the unligatable open circular plasmid can be converted to 3 '-hydroxyl, 5 '-phosphate nicked plasmid.
  • Any method for converting unligatable open circular plasmid to 3 '-hydroxyl, 5 '-phosphate nicked plasmid may be used.
  • Other methods may be provided using the many enzymes and methods known in the art of DNA repair (Friedberg et al. DNA Repair and Mutagenesis, ASM Press, 1995).
  • Second Enzymatic Reaction Conversion of 3 '-hydroxyl, 5'-phosphate nicked plasmid to relaxed covalently closed circular plasmid.
  • second reaction the 3 '-hydroxyl, 5 '-phosphate nicked plasmid is converted in vitro to relaxed covalently closed circular plasmid. This is accomplished by incubation with a DNA ligase in the presence of DNA ligase nucleotide cofactor.
  • Third Enzymatic Reaction Conversion of relaxed covalently closed circular plasmid to negatively supercoiled plasmid.
  • the relaxed covalently closed circular plasmid is converted in vitro to negatively supercoiled plasmid. This is accomplished by incubation with a DNA gyrase in the presence of DNA gyrase nucleotide cofactor (usually ATP).
  • DNA gyrase nucleotide cofactor usually ATP
  • the repair of open circular plasmid in a plasmid preparation has not been previously demonstrated experimentally.
  • the nature of the DNA damage in open circular plasmid in plasmid preparations has not been investigated in the literature. To date, no one has experimentally demonstrated that this open circular plasmid can be converted to supercoiled plasmid in vitro. This is the first demonstration that such open circular plasmid can be converted in vitro.
  • the conversion of open circular plasmid to supercoiled plasmid may be nearly quantitative. Nearly all of the open circular plasmid may be converted to supercoiled plasmid.
  • the three enzymatic conversion reactions are preferably performed simultaneously in a single combined incubation, using an enzyme mixture.
  • the enzyme mixture may comprise DNA polymerase, DNA ligase, and DNA gyrase. This mixture may further comprise one or more 3' deblocking enzymes. This mixture may further comprise one or more 5' deblocking enzymes.
  • the enzyme mixture may comprise 3 '-phosphatase, polynucleotide kinase, DNA ligase, and DNA gyrase.
  • the three conversion reactions may also be performed sequentially in the order: first reaction, second reaction, and third reaction.
  • the first and second reactions may be performed simultaneously, followed by the third reaction.
  • the first reaction may be performed, followed by the second and third reactions simultaneously.
  • the optimal incubation conditions such as temperature or pH or buffer conditions, differ for the enzymes used herein, it may be advantageous to perform the conversion reactions sequentially.
  • the conversion reactions may be performed with intermediate purification of plasmid between conversion reactions. A disadvantage of such intermediate purification embodiments is that a substantial amount of plasmid may be lost in the intermediate purification.
  • the conversion reactions are performed without intermediate purification of plasmid.
  • relaxed covalently closed circular plasmid may have the same bioactivity as supercoiled plasmid.
  • the third reaction with DNA gyrase may be omitted. If the second reaction with DNA ligase is performed in the presence of an intercalating agent, then removal of the intercalating agent after ligation will result in negatively supercoiled plasmid.
  • the second reaction is performed in the absence of an intercalating agent, due to the carcinogenic nature of intercalating agents.
  • the conversion reactions will convert at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of open circular plasmid in the plasmid solution to supercoiled plasmid.
  • Enzymes 3 '-Phosphatase and polynucleotide kinase enzymes from any source may be used provided that they are active on open circular plasmid substrate.
  • Polynucleotide kinase and 3 '-phosphatase enzyme activities are sometimes found on a single polypeptide in some organisms, known as PNKP.
  • PNKP has been characterized in numerous organisms, including rats, human, bovine, plasmodium, S. pombe, and mouse (Karimi-Busheri et al., Nucl. Acids Res. 26:4395-4400, 1998).
  • 3 '-Phosphatase with no associated polynucleotide kinase activity has been characterized in Saccharomyces cereviseae and Arabidopsis thaliana (Vance et al., J. Biol. Chem. 276:15073-15081, 2001).
  • Polynucleotide kinase with no associated 3 '-phosphatase could potentially be obtained by mutation of PNKP.
  • the polynucleotide kinase and 3 '-phosphatase enzymes may be present on separate proteins, but preferably are present on the same protein (PNKP).
  • a preferred source of PNKP is human. DNA polymerases from any source may be useful: e.g.
  • Klenow DNA polymerase eubacterial DNA polymerases, phage DNA polymerases, viral DNA polymerases, eukaryotic DNA polymerases, archaebacterial DNA polymerases, and genetically mutated versions thereof.
  • the DNA polymerase does not have substantial strand displacing activity on open circular plasmid.
  • a preferred DNA polymerase has both 3 '-5' and 5 '-3' exonuclease activities, such as DNA polymerase I from some sources. DNA polymerase I is likely found in many organisms.
  • a preferred source of DNA polymerase I is E. coli.
  • DNA ligase from any source may be used, provided that it is capable of ligating 3'-hydroxyl, 5'-phosphate nicks.
  • DNA ligase is found in many organisms.
  • DNA ligases from bacteriophages, viruses, eukaryotes, and archaebacteria usually require adenosine triphosphate (ATP) as the nucleotide cofactor.
  • DNA ligases from eubacteria, such as E. coli usually require nicotinamide adenine dinucleotide (NAD) as the cofactor.
  • NAD nicotinamide adenine dinucleotide
  • the DNA ligase requires ATP cofactor.
  • a preferred source of DNA ligase is bacteriophage T4.
  • DNA gyrase from any source can be used, provided that it converts relaxed covalently closed circular plasmid to supercoiled plasmid.
  • DNA gyrase is found in eubacteria and some archeabacteria.
  • DNA gyrase converts relaxed covalently closed circular plasmid to negatively supercoiled plasmid in the presence of ATP or an equivalent nucleotide.
  • a preferred source of DNA gyrase is E. coli.
  • DNA gyrase Vibrio cholera, which is reported to be unable to catalyze the reverse reaction (Mukhopadhyay et al., Biochemical J. 280:797-800, 1991).
  • Another useful source of DNA gyrase could be mycobaterium smegmatis, which is reported to have stronger decatenase activity.
  • the incubation with DNA gyrase is preferably performed substantially in the absence of topoisomerase I.
  • Reverse DNA gyrase may be used instead of DNA gyrase.
  • Reverse DNA gyrase is found in many thermophilic bacteria. Reverse DNA gyrase converts relaxed covalently closed circular plasmid to positively supercoiled plasmid.
  • DNA gyrase reverse DNA gyrase
  • DNA gyrase is employed, because negatively supercoiled plasmid is known to be biologically active in human cells.
  • Repair Enzymes and Accessory Proteins The repair of single strand breaks in double stranded DNA is an essential function of the DNA repair system of all living organisms. Numerous repair enzymes and accessory proteins are known which facilitate the repair of single strand breaks. Such enzymes and accessory proteins could be used to accelerate or improve the conversion of unligatable open circular plasmid to covalently closed circular plasmid.
  • Non-limiting examples of other proteins / enzymes of potential use in repairing single stranded breaks in open circular plasmid may include protein HU, XRCC1, RNase H, DNA glycosylases, damage-specific endonucleases (e.g. UvrABC), and enzymes involved in single strand break repair, base excision repair, nucleotide excision repair, or mismatch repair (Friedberg et al. DNA Repair and Mutagenesis, ASM Press, 1995).
  • nucleotide Cofactor Regeneration Several enzymes used herein require nucleotide cofactors. DNA gyrase and polynucleotide kinase require ATP for activity, generating ADP as the nucleotide byproduct of the cofactor. DNA ligase requires ATP (or NAD) for activity, generating AMP (or NMP) as the nucleotide by-product of the cofactor. It will be appreciated that equivalent cofactors may potentially be used (e.g. dATP). Optionally, the nucleotide by- product of the cofactor may be enzymatically converted back to nucleotide cofactor during one or more of the reactions, thus, helping to maintain a constant concentration of nucleotide cofactor.
  • ADP generated by DNA gyrase may be converted back to ATP using a kinase enzyme and a high energy phosphate donor (i.e., the kinase substrate).
  • the preferred kinase enzyme and phosphate donor are pyruvate kinase and phosphoenolpyruvate (PEP).
  • Other kinase and high energy phosphate donors may include creatine kinase and creatine phosphate, and acetate kinase and phosphoacetate.
  • ADP generated by polynucleotide kinase may be converted back to ATP using a kinase enzyme and a high energy phosphate donor.
  • AMP generated by DNA ligase may be converted back to ATP using a mixture of adenylate kinase, kinase enzyme, and high energy phosphate donor.
  • the cofactor for DNA ligase is NAD
  • the nucleotide byproduct NMP may be converted back to NAD during the second reaction by the enzyme nicotinamide adenylyltransferase.
  • AMP generated by this enzyme may be converted back to ATP as described. Pyrophosphate is generated as a by-product of the DNA ligase and the DNA polymerase reactions.
  • inorganic pyrophosphatase may be included during the incubation with DNA ligase and/or DNA polymerase, to hydrolyze pyrophosphate to phosphate.
  • the use of enzymes for regenerating nucleotide cofactor from their nucleotide by-product is optional.
  • Example 3 demonstrates a non-limiting embodiment using ATP regeneration.
  • An optional additional in vitro enzymatic reaction with one or more exonucleases may be performed to reduce linear chromosomal DNA contamination in the plasmid solution.
  • the linear chromosomal DNA may be reacted with one or more exonucleases, wherein said exonucleases have at least some substrate selectivity in preferentially degrading linear chromosomal DNA substrate versus covalently closed circular plasmid substrate, whereby at least some linear chromosomal DNA is degraded.
  • the exonuclease reaction is preferably performed without substantially hydrolyzing covalently closed circular plasmid.
  • the exonuclease reaction may also advantageously degrade open circular plasmid which is remaining after the second reaction.
  • selectivity of the exonucleases need not be absolute. Most exonucleases lack absolute substrate specificity. A loss of plasmid due to lack of absolute substrate specificity by an exonuclease may be necessary to achieve desired reduction in chromosomal DNA. The selection of the exonuclease(s) depends on when the reaction is performed.
  • the linear chromosomal DNA may be reacted with one or more exonucleases, wherein said exonucleases have at least some substrate selectivity in preferentially degrading linear chromosomal DNA substrate versus open circular and covalently closed circular plasmid substrates, whereby at least some linear chromosomal DNA is degraded.
  • This exonuclease reaction is preferably performed without substantially hydrolyzing open circular and covalently closed circular plasmid.
  • Non-limiting examples of such exonucleases may include exonuclease I, lambda exonuclease, exonuclease V, exonuclease VE, exonuclease VEI, exonuclease T (RNase T), rec f, or combinations thereof.
  • exonucleases may be conveniently used concurrently with all the conversion reactions.
  • deblocking enzymes which are also exonucleases may potentially serve a dual function of hydrolyzing chromosomal DNA. Some plasmid (such as open circular plasmid or closed circular plasmid) may be degraded due to a lack of absolute exonuclease substrate specificity.
  • the optional exonuclease reaction is preferably performed concurrently with the conversion reactions, preferably using exonuclease V, preferably with low helicase activity.
  • a preferred source of exonuclease V is M. luteus.
  • ADP generated by exonuclease V may be converted back to ATP as described.
  • Example 4 demonstrates non-limiting embodiments using concurrent exonuclease digestion.
  • the linear chromosomal DNA may be reacted with one or more exonucleases, wherein said exonucleases have at least some substrate selectivity in preferentially degrading linear chromosomal DNA substrate versus covalently closed circular plasmid substrate, whereby at least some linear chromosomal DNA is degraded.
  • This exonuclease reaction is preferably performed without substantially hydrolyzing covalently closed circular plasmid.
  • Non-limiting examples of such exonucleases may include exonuclease I, exonuclease El, exonuclease V, exonuclease VE, exonuclease VEI, lambda exonuclease, T7 exonuclease, T5 exonuclease, exonuclease T, RecJf, or combinations thereof.
  • DNA polymerase I may be used as an exonuclease in the absence of dNTP substrates. Such exonucleases may be conveniently used subsequent to the conversion reactions.
  • Some covalently closed circular plasmid may be degraded by the exonuclease reaction due to a lack of absolute exonuclease substrate specificity.
  • the conversion of open circular plasmid to covalently closed circular plasmid in the conversion reactions will usually not be 100%, resulting in remaining open circular plasmid after the second reaction.
  • this exonuclease reaction may also advantageously degrade remaining open circular plasmid. This is accomplished using an exonuclease which degrades open circular plasmid, for example using exonuclease El.
  • DNA polymerase and dNTPs are present during this exonuclease digestion using exonuclease IE, then the concentration of exonuclease El should be adjusted appropriately to effect digestion of linear double stranded chromosomal DNA.
  • DNA polymerase and/or other enzymes used in the conversion reactions may optionally be inactivated prior to the subsequent exonuclease digestion, such as by heat inactivation.
  • Example 5 demonstrates a non-limiting embodiment using subsequent exonuclease digestion.
  • the amount of plasmid degraded during the exonuclease reaction may depend on several factors, such as substrate specificity of the exonuclease(s), whether the exonucleases are used to remove remaining open circular plasmid after the second reaction and the amount of remaining open circular plasmid, and reaction conditions (enzyme concentrations, incubation times, etc).
  • the plasmid loss is preferably not substantial; however, in some cases, the loss may be substantial. For example, plasmid loss may be substantial if a large amount of open circular plasmid is remaining after the second reaction, and this remaining open circular plasmid is degraded by exonuclease.
  • the exonuclease reaction may be performed using one or more single stranded DNA exonucleases, such as exonuclease I.
  • exonuclease I single stranded DNA exonucleases
  • double stranded linear chromosomal DNA could be converted to single stranded form by a brief denaturation step after the second reaction and prior to exonuclease digestion.
  • the exonuclease reaction may be performed using a combination of one or more single stranded DNA exonucleases and one or more double stranded DNA exonucleases.
  • One advantageous combination comprises exonuclease I and exonuclease IE.
  • one or more exonucleases is incubated concurrently with the conversion reactions (e.g. exonuclease I).
  • exonucleases is then added to further digest chromosomal DNA (e.g. exonuclease ET). Additional enzymes, such as PNKP or exonuclease IE, may be useful in converting the termini of linear chromosomal DNA to the desired phosphorylation state to facilitate exonuclease digestion.
  • plasmid may be purified prior to the exonuclease reaction. Preferably though, after a conversion reaction, plasmid is not purified prior to the exonuclease reaction.
  • exonucleases for selective hydrolysis of chromosomal DNA in combination with conversion of open circular plasmid to supercoiled plasmid works synergistically to overcome the limitations of prior art uses of exonucleases.
  • Prior exonuclease digestion methods for removing chromosomal DNA fall into two categories.
  • exonucleases hydrolyze both chromosomal DNA and open circular plasmid.
  • open circular plasmid is degraded.
  • exonucleases hydrolyze only chromosomal DNA, leaving supercoiled and open circular plasmid intact.
  • the disadvantage of this approach is that open circular plasmid must be removed by subsequent purification.
  • the combination of exonuclease digestion of chromosomal DNA and conversion of open circular plasmid to supercoiled plasmid overcomes these disadvantages of the prior art.
  • a single incubation could potentially produce high purity supercoiled plasmid with low levels of contaminating chromosomal DNA without significant loss of plasmid.
  • the optional exonuclease reaction may be especially advantageous for low copy plasmids, which tend to have a higher percentage of chromosomal DNA contamination than high copy plasmids.
  • the optional exonuclease reaction may be useful in combination with any method which converts open circular plasmid to supercoiled plasmid.
  • a ribonuclease could be used to hydrolyze residual RNA.
  • Ribonuclease incubation may be performed as a separate incubation or simultaneously with one or more conversion reactions.
  • a preferred ribonuclease is ribonuclease I.
  • undesired plasmid may be removed by selective restriction endonuclease digestion. If two or more plasmids are present in a plasmid solution, usually only one plasmid is the desired product. For example, a host cell may contain two different plasmids. Alternatively, two different plasmids could be generated from one plasmid by incubation with a recombinase.
  • the resulting selectively linearized undesired plasmid could be further hydrolyzed by incubation with an exonuclease(s).
  • Temperature may advantageously be used as an on/off switch of enzyme activity.
  • the conversion reactions may be performed at 37°C using E. coli enzymes. After completing the conversion reactions, the temperature could be increased to 60°C for selective degradation of chromosomal DNA using thermophilic exonuclease(s), such as exonuclease I and/or IE.
  • thermophilic exonuclease(s) such as exonuclease I and/or IE.
  • the E. coli enzymes are likely to be inactive.
  • the thermophilic exonuclease(s) may be poorly active, and thus not interfere with the conversion reactions.
  • Catenation DNA gyrase is known to reversibly catalyze the formation of catenanes (Kreutzer, Cell 20:245-254, 1980; Krasnow, J. Biol. Chem. 257:2687-2693, 1982).
  • a catenane is formed by interlocking of two or more plasmid molecules, forming dimers or multimers. The formation of catenanes may be undesirable for gene transfer due to their larger molecular size.
  • the DNA gyrase incubation is performed to avoid or to minimize formation of catenanes. This may be accomplished by appropriate selection of buffer composition, such as the spermidine concentration or salt concentration, as taught in prior art.
  • the buffer composition could be selected such that the amount of catenanes in the plasmid solution would be reduced by the DNA gyrase incubation. Conversely, for applications in which catenanes are desirable, the buffer composition could be selected so that the amount of catenanes would be increased by the DNA gyrase incubation. In the examples, no significant catenation was observed. At the highest plasmid concentration in Example 1 of 1.5 ⁇ g/ ⁇ l, no significant catenation was observed. Based on visual inspection of agarose gels in the examples, it is estimated that the amount of catenane formation is less than approximately 1% to 5% of total plasmid.
  • the amount of catenane formation resulting from the third reaction is less than 1%, less than 5%, less than 10%, less than 15%, or less than 20% of the total plasmid; preferably, without the use of a potent decatenase. If catenane formation does occur to an undesirable extent, then catenane formation could be reduced by several possible methods.
  • the DNA gyrase reaction could be performed at a lower plasmid concentration or using a buffer composition that minimizes plasmid aggregation.
  • a DNA gyrase with stronger decatenase activity could be used, such as Mycobacterum smegmatis DNA gyrase.
  • Catenation could be reduced or eliminated by an optional additional incubation with a potent decatenase enzyme, such as topoisomerase IE or preferably topoisomerase IV.
  • a potent decatenase enzyme such as topoisomerase IE or preferably topoisomerase IV.
  • the incubation with a potent decatenase is preferably performed simultaneously with the DNA gyrase reaction, but could be performed after the DNA gyrase reaction. Both potent decatenases relax supercoiled plasmid at a slow rate. Therefore, the potent decatenase is preferably used at a minimal concentration, to effect decatenation and to minimize supercoiled relaxation. ADP generated by the potent decatenases could be converted back to ATP as described earlier. Preferably though, incubation with a potent decatenase is not performed.
  • the resulting plasmid may be used directly in some applications without further purification.
  • additional plasmid purification may be desirable, for example to remove the buffer salts, enzymes, nucleotides, or possibly exonuclease hydrolysis products. This can be accomplished by any method, such as organic solvent extraction, chromatography, precipitation, ultrafiltration, ultracentrifugation, electrophoresis, or combinations thereof.
  • the additional purification may also remove residual open circular plasmid.
  • the additional purification may also remove residual linear chromosomal DNA.
  • the recovered supercoiled plasmid will likely be a mixture of supercoiled plasmid produced using the conversion reactions and supercoiled plasmid originally present in the cleared lysate.
  • plasmid from a cleared lysate is purified chromatographically prior to the conversion reactions. After the conversion reactions, the plasmid product is purified chromatographically as a final "polishing" procedure.
  • the preferred chromatographic method is anion exchange, before and after the conversion reactions.
  • Commercially available anion exchange column for plasmid purification may be useful (Qiagen, Macherey-Nagel).
  • the same chromatographic column is used before and after the conversion reactions, preferably an anion exchange column.
  • Applications for the recovered supercoiled plasmid may include transformation into recipient competent cells, such as tissue culture or whole animals, and especially for human therapeutic use.
  • recipient competent cells such as tissue culture or whole animals
  • the conversion reactions are used in combination with the optional exonuclease reaction, the final plasmid product may have a high percentage of supercoiled plasmid and a low percentage of chromosomal DNA contamination.
  • one or more of the enzymes could be covalently attached to a solid support and packed in a column, producing an immobilized enzyme column.
  • An immobilized enzyme column could be made for each enzyme in the method separately; alternatively, a single immobilized enzyme column could contain a mixture of enzymes to convert unligatable open circular plasmid to supercoiled plasmid. Plasmid solution could be pumped through the column, or series of columns, converting unligatable open circular plasmid to supercoiled plasmid. Column eluate could be recycled through the column(s) as needed until most of the unligatable open circular plasmid is converted to supercoiled plasmid.
  • An immobilized enzyme column could be used multiple times to prepare multiple plasmids with appropriate washing before reuse.
  • the enzymes are not attached to a solid support and are free in solution.
  • large quantities of enzymes may be needed.
  • the enzyme(s) must be separated from the plasmid. This may be accomplished by using affinity chromatography (e.g. if the enzymes have an affinity tag) or classical chromatography (e.g. anion or cation exchange or dye ligand). Loss of enzyme activity during incubation is preferably minimized.
  • one or more enzymes may be thermostable and derived from a thermophilic organism.
  • some or all of the enzymes could be derived from a thermophilic prokaryote, such as Bacillus stearothermophilus, or a thermophilic eukaryote, such as Thermomyces lanuginosus.
  • the incubations with thermostable enzyme could be performed at temperatures between about 45°C and 75°C.
  • Thermostable enzymes would likely maintain most of their activity during the incubation, optionally allowing reuse for subsequent incubations if desired.
  • Reactions Preferably Not Performed
  • Most plasmid preparations contain a mixture of supercoiled and open circular plasmid prior to the conversion reactions. After preparing a cleared lysate, it is preferable to preserve the supercoiled plasmid prior to and during the conversion reactions. Therefore, additional reactions which work against this objective are preferably not performed. After preparing a cleared lysate, the conversion reactions are preferably performed without prior purposeful in vitro conversion of supercoiled plasmid to an undesired form.
  • Undesired forms include linear, open circular, relaxed covalently closed circular, replicated daughter plasmids (partial or complete), single stranded circular, triple stranded, single-strand invasion, or Holliday structure forms.
  • After preparing a cleared lysate it is preferable to preserve the open circular plasmid so that it may be quantitatively converted to supercoiled plasmid. Therefore, additional reactions which work against this objective are preferably not performed.
  • the first and second reactions are preferably performed without prior purposeful in vitro conversion of open circular plasmid to an undesired form.
  • Undesired forms include linear, single stranded circular, triple stranded, single- strand invasion, in vitro replicated daughter plasmids (partial or complete), Holliday structure forms, or forms with impaired ability to be subsequently converted to covalently closed circular plasmid.
  • the first and second reactions are preferably performed without prior purposeful separation of supercoiled plasmid from the open circular plasmid.
  • the following embodiments may be especially advantageous.
  • the cleared lysate usually comprises supercoiled plasmid in addition to open circular plasmid.
  • the supercoiled plasmid is preferably not purposefully modified prior to the first reaction.
  • Purposeful modification is usually a quantitative conversion, in which most of the material is converted to a different form.
  • supercoiled plasmid from the cleared lysate is not purposefully converted to open circular plasmid, for example by intentional free radical nicking.
  • supercoiled plasmid is not purposefully converted to relaxed covalently closed circular plasmid, for example by intentional incubation with topoisomerase I.
  • supercoiled plasmid (or open circular plasmid) is not purposefully converted to linear form, for example by restriction digestion.
  • open circular plasmid in the plasmid solution is not purposefully converted to single stranded circular DNA, for example by heat or alkali.
  • open circular plasmid is not purposefully separated from supercoiled plasmid.
  • the conversion reactions are performed without purposeful in vitro plasmid replication and without prior purposeful in vitro plasmid replication.
  • "In vitro plasmid replication” is defined herein as enzymatic production of daughter plasmid molecules (either partial or complete synthesis) from a parent plasmid in vitro. Partial production of daughter molecules on some plasmids begins with initiation of new strand synthesis and produces a theta structure as viewed with an electron microscope. Partial production of daughter molecules by rolling circle replication results in production of single stranded molecules from the parent plasmid.
  • DNA polymerase may generate a small amount of displaced single stranded DNA by strand displacement as an unintentional side reaction of DNA repair of open circular plasmid, not as intentional plasmid replication.
  • flaps may potentially be repaired using a flap endonuclease.
  • An example of in vitro plasmid replication is described by Funnel et al. (J. Biol. Chem. 261:5616-5624, 1986).
  • in vitro plasmid replication is not performed after the conversion reactions.
  • the nucleotide sequence of the plasmid is not modified.
  • the conversion reactions are performed without an in vitro incubation, or prior in vitro incubation, with a primase enzyme or an RNA polymerase enzyme, which produces primers for synthesis of daughter strands of plasmid.
  • the plasmid solution may further comprise purposefully in vitro synthesized open circular plasmid.
  • the plasmid solution does not comprise open circular plasmid which was purposefully synthesized by an in vitro method.
  • the conversion reactions are performed without purposeful in vitro synthesis of open circular plasmid, for example from nucleic acid which is not open circular plasmid.
  • the conversion reactions are performed without increasing the total amount of plasmid in vitro, where conversion of gapped plasmid in the plasmid solution to closed circular plasmid is not considered increasing the amount of total plasmid.
  • the conversion reactions are performed without increasing in vitro the total number of plasmid molecules.
  • the conversion reactions may be performed so that the total amount of plasmid is substantially unchanged.
  • a substantial amount of plasmid may be lost, such as potentially in the optional exonuclease reaction.
  • the conversion reactions are performed so that the amount of supercoiled plasmid after the conversion reactions is increased from the starting amount of supercoiled plasmid immediately prior to the conversion reactions.
  • the conversion reactions are performed so that the percentage of supercoiled plasmid is increased from the starting percentage of supercoiled plasmid immediately prior to the conversion reactions. Preferably, this is accomplished without separation of open circular plasmid from supercoiled plasmid prior to completing the second reaction.
  • the conversion reactions are performed in a manner to minimize or avoid in vitro recombination events.
  • the conversion reactions are preferably performed in the absence of RecA protein or in the absence of single stranded DNA binding protein.
  • the conversion reactions are performed without purposeful conversion of plasmid to triple stranded forms, Holliday structures, or other strand invasion forms, and/or without prior such in vitro conversion.
  • the conversion reactions are performed using purified enzymes. This can be accomplished by using recombinant enzymes purified by chromatography.
  • the conversion reactions are not performed using a crude extract as a source of enzyme, such as a cell lysate.
  • the conversion reactions are performed without incorporating modified nucleotide analogs into the plasmid.
  • the open circular plasmid in the plasmid solution consists of (i) open circular plasmid which existed in host cells immediately prior to lysis, or (ii) supercoiled plasmid in host cells which was unintentionally converted to open circular plasmid in the preparation of the cleared lysate, or (iii) supercoiled plasmid in the cleared lysate which was unintentionally converted to open circular plasmid after further plasmid purification from other host cell components, or (iv) combination thereof.
  • Unintentional conversion is the consequence of the inherent instability of supercoiled plasmid to DNA damage.
  • essentially all of the plasmid in the plasmid solution was synthesized by the host cells.
  • unintentional plasmid modification may occur. This may result from enzyme impurities.
  • nuclease contamination may convert some supercoiled plasmid to open circular plasmid.
  • Unintentional conversion may also result from the side reactions due to inherent activities of the enzymes used. Several examples illustrate this point.
  • (1) The optional exonuclease reaction may hydrolyze some plasmid due to lack of absolute substrate selectivity.
  • AP endonuclease may convert some supercoiled plasmid to open circular plasmid, if the supercoiled plasmid contains an abasic site. This conversion is not considered purposeful, since the purpose of the AP endonuclease is the repair of open circular plasmid.
  • a preferred enzyme composition comprises DNA polymerase, DNA ligase, and DNA gyrase.
  • the preferred composition may further comprise one or more 3' deblocking enzymes.
  • the preferred composition may further comprise one or more 5' deblocking enzymes.
  • a preferred enzyme composition for the alternate mode comprises polynucleotide kinase, 3 '-phosphatase, DNA ligase, and DNA gyrase.
  • the enzyme composition may further comprise one or more of the following enzymes: (1) kinase enzyme to regenerate nucleotide cofactor, (2) one or more exonucleases to hydrolyze residual chromosomal DNA, (3) inorganic pyrophosphatase, (4) ribonuclease, and (5) topoisomerase IV.
  • the enzyme composition does not comprise additional enzymes which result in (i) in vitro plasmid replication and (ii) conversion of single stranded circular DNA to open circular DNA without using a synthetic primer. Examples of such additional enzymes include primase, RNA polymerase, and single stranded DNA binding protein.
  • the enzyme composition does not comprise substantial topoisomerase I contamination.
  • the enzymes of the composition could be produced using recombinant DNA technology as genetic fusions with affinity fusion protein tags, such as polyhistidine, to facilitate purification.
  • the enzymes could be purified to decrease endotoxin contamination to low levels.
  • the enzyme reagents could be supplied in dry lyophilized form, or as an aqueous solution (e.g. buffered 50% glycerol solution).
  • Advantages over Prior Art The present invention offers three potential fundamental advantages over prior art methods: (1) increased yield of supercoiled plasmid, (2) uniformly highly supercoiled state, and (3) one universal procedure for all plasmids. These advantages are discussed further. The above methods differ in a fundamental manner from prior art methods for purifying supercoiled plasmid.
  • Prior art methods are based on excluding open circular plasmid from the final plasmid preparation.
  • the invention is based on including derivatives of open circular plasmid in the final plasmid preparation, by enzymatically converting open circular plasmid to supercoiled plasmid.
  • nearly all of the open circular plasmid can be converted to supercoiled plasmid.
  • one advantage over prior art methods is increased supercoiled plasmid yield.
  • the inventor has observed substantially no loss of plasmid in the conversion reactions. This is illustrated in Examples 1 and 2.
  • prior art methods are based on separation, which involves loss of plasmid.
  • This •method may be especially useful for bulk scale plasmid preparations, which tend to have a higher percentage of open circular plasmid due to longer processing times.
  • the above methods provide a solution to a previously unrecognized problem in the art of plasmid preparation - the extent of supercoiling.
  • the extent of supercoiling of plasmid can vary from batch to batch and under different host cell growth conditions.
  • the extent of supercoiling may have an effect on the biological activity of the plasmid. For example, a plasmid preparation which has a low extent of supercoiling may be less bioactive than desired.
  • the extent of supercoiling of plasmid in bacteria is not at its thermodynamic maximum (Cullis et al., Biochemistry 31:9642- 9646, 1992). This is due to topoisomerase I which relaxes supercoiled plasmid in the bacterial host.
  • the extent of supercoiling in vivo is an equilibrium effect between DNA gyrase and topoisomerase I. Occasionally, the extent of supercoiling in a host may be far below normal. This poorly supercoiled plasmid could occur during the fermentation of host cells, possibly due to nutrient starvation, cell death, low ATP energy charge, or other effect. This previously unrecognized problem may be solved by DNA gyrase incubation in the third reaction.
  • the DNA gyrase incubation could increase the extent of supercoiling to its maximum thermodynamic limit.
  • the increased supercoiling of the plasmid could create a more condensed molecule with potentially greater transformability.
  • the DNA gyrase incubation could convert plasmid (including supercoiled plasmid from the cleared lysate) to a more uniformly highly supercoiled and condensed state.
  • Enzyme concentrations and incubation times may be the same for all plasmids, regardless of plasmid size, plasmid GC content, plasmid DNA sequence, percent supercoiled plasmid in the plasmid solution, and percent of chromosomal DNA contamination.
  • the details of the procedure would not need to be optimized for each individual plasmid.
  • a single universal procedure may work well for all plasmids.
  • prior art methods usually require optimization for each individual plasmid, in order to maximize the separation of supercoiled from open circular plasmid, while minimizing loss of supercoiled plasmid.
  • chromatographic purification of supercoiled plasmid usually requires optimization of the gradient and sample load amount for each individual plasmid.
  • a further advantage may be to ensure consistent and reproducible proportions of supercoiled plasmid in the final plasmid preparation, reducing batch to batch variation.
  • DNA gyrase, DNA ligase, DNA polymerase, polynucleotide kinase, and 3 '-phosphatase have never been applied in the field of plasmid purification. The use of these enzymes breaks new ground in the art of plasmid preparation.
  • T4 DNA ligase and human PNKP were produced as fusion proteins with glutathione-S-transf erase (GST) affinity tag as follows.
  • the genes coding for these enzymes were amplified by the polymerase chain reaction.
  • the genes were cloned into pGEX, a commercially available expression vector (Amersham) so that the GST affinity tag was fused to the amino terminus of the enzyme.
  • the fusion proteins were purified on glutathione-agarose according to the manufacturer's instructions. These fusion proteins are denoted GST-T4 DNA ligase and GST-PNKP.
  • E. coli DNA gyrase was obtained from John Lines Ltd. E.
  • coli DNA polymerase I phage T4 DNA polymerase, phage lambda exonuclease, phage T7 exonuclease (gene 6), E. coli exonuclease I, and E. coli exonuclease IE were obtained from New England Biolabs.
  • E. coli endonuclease IV was obtained from Epicentre.
  • M. luteus exonuclease V was obtained from USB Corp. Enzyme concentrations were not necessarily optimized in the following examples. For instance, the first part of Example 1 was repeated using one- tenth the amount of GST-T4 DNA ligase with substantially the same result.
  • coli host was prepared using the alkaline lysis method, followed by further purification to remove RNA and protein. Agarose gel electrophoresis showed approximately 30% open circular plasmid, 70% supercoiled plasmid, and some residual chromosomal DNA was likely present.
  • This plasmid preparation denoted p4kb, was used in the subsequent examples.
  • EXAMPLE 1 Preferred Mode A 10 ⁇ l reaction volume contained 5 ⁇ g p4kb plasmid, 35 mM Tris-HCl (pH
  • DNA ligase 0.2 units DNA polymerase I, 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, and 200 ⁇ M dTTP. This reaction was incubated at 37°C for 2 hours. After incubation, the plasmid was analyzed by agarose gel electrophoresis. The gel showed a high yield of supercoiled plasmid, confirming conversion of most of the open circular plasmid to supercoiled plasmid. By visual inspection of the stained gel, it is estimated that about 80% to 85% of open circular plasmid was converted to supercoiled form. Based on flourometry analysis, the total amount of plasmid measured before and after the reaction was the same.
  • a 2-hour incubation using 5 ⁇ g p4kb and 0.2 units T4 DNA polymerase (instead of DNA polymerase I) resulted in about 40% conversion.
  • EXAMPLE 2 Preferred Mode + 3' deblocking enzyme A 10 ⁇ l reaction volume contained 5 ⁇ g ⁇ 4kb plasmid, 35 mM Tris-HCl (pH 7.5), 25 mM KCl, 4 mM MgCl 2 , 2 mM dithiothreitol, 1.8 mM spermidine, 1 mM ATP,
  • DNA ligase 0.2 units DNA polymerase I, 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, 200 ⁇ M dTTP, and 0.5 units exonuclease ET. This reaction was incubated at
  • EXAMPLE 3 Preferred Mode + ATP regeneration
  • a 10 ⁇ l reaction volume contained 5 ⁇ g p4kb plasmid, 35 mM Tris-HCl (pH 7.5), 25 mM KCl, 4 mM MgCl 2 , 2 mM dithiothreitol, 1.8 mM spermidine, 1 mM ATP, 6.4% glycerol, 0.1 mg/ml bovine serum albumin, 2.5 units DNA gyrase, 2.8 ⁇ g GST-T4 DNA ligase, 0.2 units DNA polymerase I, 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, 200 ⁇ M dTTP, 0.05 units creatine kinase (Sigma C3755), and 1 mM creatine phosphate.
  • This reaction was incubated at 37°C for 2 hours. After incubation, the plasmid was analyzed by agarose gel electrophoresis. The gel showed high purity supercoiled plasmid, confirming conversion of most of the open circular plasmid to supercoiled plasmid. By visual inspection of the stained gel, it is estimated that about 75% to 80% of open circular plasmid was converted to supercoiled form.
  • EXAMPLE 5 Preferred Mode + Subsequent Exonuclease Digestion
  • a 20 ⁇ l reaction volume contained 5 ⁇ g p4kb plasmid, 35 mM Tris-HCl (pH 7.5), 25 mM KCl, 4 mM MgCl 2 , 2 mM dithiothreitol, 1.8 mM spermidine, 1 mM ATP, 6.4% glycerol, 2.5 units DNA gyrase, 2.8 ⁇ g GST-T4 DNA ligase, 0.2 units DNA polymerase I, 200 ⁇ M dATP, 200 ⁇ M dGTP, 200 ⁇ M dCTP, and 200 ⁇ M dTTP.
  • This reaction was incubated at 37°C for 2 hours. After this conversion reaction incubation, the following exonucleases were subsequently added: 0.5 ⁇ l 20 units/ ⁇ l exonuclease I, 1.0 ⁇ l 10 units/ ⁇ l T7 exonuclease, and 1.0 ⁇ l 10 units/ ⁇ l exonuclease El. The reaction was incubated an additional 2 hours at 37°C. After incubation, the plasmid was analyzed by agarose gel electrophoresis. The stained gel showed only supercoiled plasmid, with no visible open circular plasmid. Based on flourometry, the loss of DNA in the subsequent exonuclease incubation was about 12%.
  • This DNA loss is likely a loss of both linear chromosomal DNA and residual open circular plasmid.
  • This residual open circular plasmid remaining after the conversion reactions, is subsequently degraded by both exonuclease El and T7 exonuclease.
  • This exonuclease mixture used at this concentration and duration, may reduce linear chromosomal DNA contamination by 50 fold. Based on visual inspection of the stained gel, no significant degradation of supercoiled plasmid was observed by this subsequent exonuclease incubation.

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Abstract

Selon un mode de réalisation de l’invention, il est prévu un procédé de fabrication de plasmagène à partir de cellules hôtes contenant le plasmagène, consistant : (a) à prévoir une solution plasmagène composée de plasmagène circulaire ouvert à ligature impossible ; (b) à mettre en réaction le plasmagène circulaire ouvert à ligature impossible avec une ou plusieurs enzymes et des cofacteurs nucléotides appropriés, pour convertir le plasmagène circulaire ouvert à ligature impossible en plasmagène nickelé 3’-hydroxyle, 5'-phosphate ; (c) à mettre en réaction le plasmagène nickelé 3’-hydroxyl, 5'-phosphate avec un ligase ADN et un cofacteur nucléotide de ligase ADN, pour convertir le plasmagène nickelé 3’-hydroxyl, 5'-phosphate en plasmagène circulaire fermé de manière covalente relâché; et (d) à mettre en réaction le plasmagène circulaire fermé de manière covalente relâché avec un gyrase ADN et cofacteur nucléotide de gyrase ADN, pour convertir le plasmagène circulaire fermé de manière covalente relâché en plasmagène surfondu de manière négative. Dans d’autres modes de réalisation, on remplace le gyrase ADN par un gyrase ADN inverse ou bien on se dispense de la réaction (d).
PCT/US2004/014946 2003-03-25 2004-05-13 Procede de fabrication de plasmagène par conversion de plasmagène circulaire ouvert en plasmagène surfondu WO2005113808A1 (fr)

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