WO2006003355A1 - Purification de molecules d'adn - Google Patents

Purification de molecules d'adn Download PDF

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Publication number
WO2006003355A1
WO2006003355A1 PCT/GB2005/000888 GB2005000888W WO2006003355A1 WO 2006003355 A1 WO2006003355 A1 WO 2006003355A1 GB 2005000888 W GB2005000888 W GB 2005000888W WO 2006003355 A1 WO2006003355 A1 WO 2006003355A1
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Prior art keywords
protein
dna
seq
plasmid
crude
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PCT/GB2005/000888
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English (en)
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Anna Victoria Hine
Richard Anthony James Darby
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Aston University
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Publication of WO2006003355A1 publication Critical patent/WO2006003355A1/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
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • 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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • the present invention relates to methods of purifying DNA molecules.
  • the invention extends to fusion proteins, and DNA molecules encoding these proteins, and to uses thereof.
  • the concept of gene therapy is to deliver one or more functional genes into a patient in order to circumvent specific genetic disorders.
  • the conveyance of these genes into the cell, and subsequently into the nucleus where they maybe expressed, can be achieved utilizing either viral or non-viral vectors.
  • many modified adenoviral, retroviral, and adeno-associated viral systems have been developed for gene therapy, as well as poxviruses and herpes simplex virus, their wide spread use is overshadowed by safety concerns. Such concerns include the immunogenic response raised in a patient, the toxicity to the patient, and the potential activation / de ⁇ activation of oncogenes and tumour suppressor genes.
  • Plasmid vectors are considered to be a safer alternative for gene therapeutic purposes. However, they are less efficient in transfection than viral vectors. It has been estimated that effective treatments would require milligram quantities of plasmid
  • plasmid based vectors DNA due to the poor cellular uptake and lack of integration into the host genome. As such large-scale bio-manufacturing must be employed if plasmid based vectors are to be widely used.
  • genomic DNA and RNA DNA
  • host proteins DNA
  • endotoxin DNA
  • plasmids have been purified from E. coli by alkaline lysis to disrupt the cells and contaminating host protein effectively removed by anion exchange chromatography (AEX). However, this does not remove genomic DNA, RNA or endotoxin.
  • Size exclusion chromatography can be employed to remove small molecular weight contaminants such as small RNA molecules and endotoxin
  • RPC reverse phase chromatography
  • SEC Size exclusion chromatography
  • RPC reverse phase chromatography
  • RPC typically employs organic solvents and toxic reagents, all of which present elevated challenges and production costs.
  • the Lactose Operon is the classical model for gene regulation. It is the model for how structural genes are transcribed or repressed depending on the conditions within the cell. When the sugar lactose is not present in the cell, a repressor protein
  • Lad is bound tightly to an operator region (lacO) of the DNA.
  • lacO operator region
  • the result of Lad binding to lacO is that the lacL gene (which codes for B-galactosidase), the lacY gene (which codes for lactose permease), and the lacA gene (which codes for transacetylase), are not transcribed.
  • the Lad repressor disassociates from the IacO operator, and the genes (lacZ, lacY, and lacA) are transcribed.
  • the Lad repressor protein comprises approximately 360 amino acids that form a homotetramer. It has five distinct fragments: four NH 2 -terminal fragments and a COOH-terminal tetrameric core.
  • the NH 2 -terminal fragments each 60 residues in length, bind in a specific manner to the IacO operator.
  • the Lad repressor monomer has four functional units: (i) the NH 2 -terminal headpiece; (ii) the hinge region; (iii) a sugar inducer binding domain; and (iv) a COOH-terminal helix.
  • the headpiece which binds to the DNA, contains a helix-turn-helix motif (HTH), which creates a small, compact globular domain that is hydrophobically rich due to two alpha helices joined by a turn to the third helix.
  • HTH helix-turn-helix motif
  • the hinge connects the DNA binding domain to the core of the Lad repressor. This segment in the absence of DNA allows the headpiece to move independently of the core of the repressor.
  • the hinge becomes ordered in an alpha helix, which causes specific interactions with the IacO operator on the DNA and it orients the headpieces.
  • the inducer binding domain core is composed of two subdomains, six- stranded parallel beta sheets sandwiched between four alpha helices.
  • the COOH-terminus contains a short segment of 11 residues followed by a COOH-terminal alpha helix that contains two leucine heptad repeats. When four of these COOH-terminal helices associate with each other, the Lad tetramer oligomerization domain is formed.
  • the Lac operon has three Lad repressor recognition sites (IacO sequences) spanning approximately 500bp. They are at the positions of three lac operator sites: (i) lacOl, (ii) lacOl, and (iii) lacO3. / ⁇ cO3 lies within the Lad gene, which is 93bp upstream of lacOl, which lies within the promoter. / ⁇ cO2 lies 401bp downstream of lacOl, within the lac Z gene. lacOl is more symmetric than / ⁇ cO2 and lacO3, and therefore it binds the tightest. The Lacl repressor binds ten times greater to the palindrome of the left half of the operator. In the Lad repressor-DNA complex, each repressor tetramer is bound to two independent, DNA operators. Therefore each Lad dimer binds to one operator site.
  • IacO sequences spanning approximately 500bp. They are at the positions of three lac operator sites: (i) lacOl, (
  • the primary site of interaction is the HTH motif of Lad, which fits tightly into the major groove of the lacO DNA.
  • Residues Leu 6, Tyr 17, GIn 18, Ser 21, Arg 22, and His 29 are close to the DNA, and their side chains most likely form the base pair interactions with the lacO operator in the major groove of the DNA.
  • the synthetic inducer molecule IPTG, binds to the Lad repressor where the NH 2 -terminal and the COOH-terminal sub-domains interface. Hence, the binding of the inducer to the Lad repressor reduces the affinity of the Lad repressor for the
  • the IPTG molecule is pseudo-symmetric, and this allows the molecule to interact with the Lad repressor molecule. Once the IPTG molecule has bound to the
  • Lad repressor the latter then undergoes a conformational change such that it unbinds itself from the lacO sequence.
  • a protein that consists of the Lad repressor protein could be used to purify a target DNA molecule that includes the lacO operator sequence due to binding therebetween.
  • the inventors constructed a fusion protein, which includes (i) the Lad repressor protein (to which the lacO of the target DNA would bind forming a protein/DNA complex) fused to (ii) a peptide linker sequence, which linker sequence can be used to bind the protein/DNA complex to a support surface.
  • the inventors further believed that knowledge of the mechanism of the inducer, IPTG, with the Lad repressor could also be harnessed to release the DNA from the protein, and hence support surface.
  • a protein comprising a Lad repressor peptide, and a His3-20 linker peptide sequence.
  • a protein comprising a Lad repressor peptide comprising an amino acid sequence substantially as set out in SEQ ID No. 12 or a functional variant thereof, and a linker peptide adapted to bind to a support surface.
  • the protein according to the first aspect comprises a fusion protein, in which the Lad repressor peptide is fused, and therefore functionally bound to the linker peptide.
  • Lad repressor peptide has four functional units: (i) a NH 2 -terminal headpiece; (ii) a hinge region; (iii) an inducer binding domain; and (iv) a COOH- terminal helix.
  • the protein according to the first or second aspect comprises at least the units (i), (ii), (iii) and
  • the N-terminal headpiece is required for binding the fusion protein to a DNA molecule comprising a lacO sequence.
  • the hinge region is required to form an alpha helix when DNA is bound to the headpiece so that the allosteric release mechanism it able to work.
  • the inducer binding domain is required for binding with IPTG, which is required to release the DNA from the fusion protein.
  • the COOH-terminal helix is required so that a Lad repressor dimer, and more preferably, a tetramer, is formed. It will be appreciated that the Lad monomer is not functional because two N-terminals are required to bind one locO sequence. A dimer is functional, but will bind only one lacO sequence.
  • the NH 2 -terminal headpiece comprises an amino acid sequence substantially as set out in SEQ ID No.14, or a functional variant thereof.
  • the hinge region comprises an amino acid sequence substantially as set out in SEQ ID No.14, or a functional variant thereof.
  • the hinge region comprises an amino acid sequence substantially as set out in SEQ ID No.14
  • the inducer binding domain comprises an amino acid sequence substantially as set out in SEQ ID No.16, or a functional variant thereof.
  • the COOH-terminal helix comprises an amino acid sequence substantially as set out in SEQ ID No.17, or a functional variant thereof.
  • the whole of the native Lad repressor is used.
  • the peptide may comprise a functional fragment of any or all of the units (i) to (iv).
  • the peptide may comprise a fragment in which the native unit (i) to (iv) has been truncated, for example, by deletion of 1, 2, 3, 4, 5, 10 or more amino acids, providing the functionality of the unit is not deleteriously impaired.
  • the peptide may comprise unit (i) in which the first few N- terminal residues have been truncated, fused to unit (ii), fused to unit (iii) and unit (iv) in which the last few C-terminal residues have been truncated, and so on.
  • the Lad repressor comprises an amino acid sequence substantially as. set out in SEQ ID No.12, or functional variant thereof.
  • the Lad repressor protein sequence is GeneID 945007 (part of the E.coli genome, accession number U00096).
  • the Lad repressor is encoded by a nucleic acid having a nucleotide sequence substantially as set out as SEQ ID No.13.
  • linker peptide we mean any suitable cross-linker, which may be adapted to bind to a support surface, and simultaneously maintain the functionality of the Lad repressor protein, such as the DNA binding function, and the inducer binding domain.
  • suitable linker peptides will be known to the skilled technician and include glutathione S-transferase, cellulose binding domain, maltose binding protein, the flag peptide, and protein A.
  • the linker peptide may comprise a His3-20 peptide sequence.
  • “His3-20 peptide sequence” we mean the linker peptide comprises between 3 and 20 hisitidine amino acid residues linked together consecutively to form a peptide.
  • His3 comprises His-His-His
  • His4 comprises His-His-His-His, and so on.
  • the linker peptide comprises a His3-15 peptide sequence, and even more preferably, a His4-10 peptide sequence.
  • the linker peptide comprises a His5-8 peptide sequence.
  • the linker peptide comprises a His6 peptide sequence, i.e. six histidine residues linked together consecutively to form a. peptide.
  • the linker peptide comprises an amino acid sequence substantially as set out in SEQ ID No.18, or a functional variant thereof.
  • the His6 cross- linker efficiently binds to a support surface used to purify the lacO DNA without any substantial conformational change or subsequent loss in function of the Lad repressor.
  • the linker peptide is encoded by a nucleic acid having a nucleotide sequence substantially as set out as SEQ ID No.19.
  • the protein according to the invention may comprise a reporter protein.
  • reporter protein we mean a protein which when expressed is detectable by means of a suitable assay procedure.
  • the reporter protein in the protein permits the localization and an estimation of protein concentration, and can also be used to determine whether any leaching occurs from the support surface during the purification process.
  • reporter proteins examples include any enzyme, which is amenable to biochemical assay, such as ⁇ -galactosidase.
  • the reporter protein may comprise a light emitting reporter protein.
  • the DNA sequence that encodes a light emitting reporter protein may code for any light emitting protein, for example, Luciferase or Green Fluorescent Protein. However, it is preferred that the DNA sequence codes for a protein that is substantially fluorescent.
  • Preferred DNA sequences that encode a light emitting reporter protein code for Green Fluorescent Protein (GFP), and light emitting derivatives thereof.
  • GFP is from the jelly fish Aequorea victoria and is able to absorb blue light and re-emits an easily detectable green light and is thus suitable as a reporter protein. GFP may be advantageously used as a reporter protein because its measurement is simple and reagent free and the protein is non-toxic.
  • Derivatives of GFP include DNA sequences encoding for polypeptide analogues or polypeptide fragments of GFP, which are able to emit light. Many of these derivatives absorb and re-emit light at wavelengths different to GFP found endogenously in Aequorea victoria.
  • the reporter protein comprises an amino acid sequence substantially as set out in SEQ ID No. 21 or functional variant thereof.
  • the reporter protein is encoded by a nucleic acid having a nucleotide sequence substantially as set out as SEQ ID No.2.0.
  • the protein in accordance with the first or second aspect may comprise NH 2 -LaCl-HiS 6 -COOH or NH 2 -His6-LacI-COOH.
  • the fusion protein comprises a GFP reporter protein
  • the protein may comprise NH 2 -LacI-His 6 -GFP-COOH, NH 2 -His6-LacI-GFP-COOH, NH 2 -His6-GFP- LacI-COOH, NH 2 -GFP-His6-LacI- COOH, NH 2 -GFP- LacI-His6-COOH, or NH 2 - LacI-GFP-His6-COOH.
  • the protein comprises an amino acid sequence substantially as set out as SEQ ID No. 23, or a functional variant thereof.
  • the fusion protein is encoded by a nucleic acid having a nucleotide sequence substantially as set out as SEQ ID No.22.
  • the protein in accordance with the first or second aspect comprises NH 2 -LaCl-X-COOH or NH 2 -X-LacI-C00H, wherein X is a suitable linker peptide.
  • nucleic acid encoding a protein according to the first or second aspect.
  • the nucleic acid may be a DNA molecule.
  • the nucleic acid has a nucleotide sequence substantially as set out as SEQ K) No.22, or a derivative or functional variant thereof.
  • the nucleic acid may be contained within a suitable vector to form a recombinant vector.
  • a vector comprising a nucleic acid according to the third aspect.
  • the vector may for example be a plasmid, cosmid or phage.
  • Such recombinant vectors are highly useful for transforming cells with the DNA molecule, for producing the fusion protein of the invention.
  • Recombinant vectors may also include other functional elements.
  • recombinant vectors can be designed such that the vector will autonomously replicate in the cell. In this case, elements which induce DNA replication may be required in the recombinant vector.
  • the recombinant vector may be designed such that the vector and recombinant DNA molecule integrates into the genome of a cell. In this case DNA sequences which favour targeted integration (e.g. by homologous recombination) are desirable.
  • Recombinant vectors may also have DNA coding for genes that may be used as selectable markers in the cloning process.
  • the recombinant vector may also further comprise a promoter or regulator to control expression of the nucleic acid as required.
  • amino acid, and nucleic acid sequences, disclosed herein may be varied to produce a derivative or a functional variant thereof.
  • derivative or “variant”, we mean that the sequence has at least 30%, preferably 40%, more preferably 50%, and even more preferably, 60% sequence identity with the amino acid/polypeptide/nucleic acid sequences of any of the sequences referred to herein.
  • An amino acid/polypeptide/nucleic acid sequence with a greater identity than preferably 65%, more preferably 75%, even more preferably 85%, and even more preferably 90% to any of the sequences referred to is also envisaged.
  • the amino acid/polypeptide/nucleic acid sequence has 92% identity, even more preferably 95% identity, even more preferably 97% identity, even more preferably 98% identity and, most preferably, 99% identity with any of the referred to sequences.
  • Calculation of percentage identities between different amino acid/polypeptide/nucleic acid sequences may be carried out as follows.
  • a multiple alignment is first generated by the ClustalX program (pairwise parameters: gap opening 10.0, gap extension 0.1, protein matrix Gonnet 250, DNA matrix IUB; multiple parameters: gap opening 10.0, gap extension 0.2, delay divergent sequences 30%, DNA transition weight 0.5, negative matrix off, protein matrix gonnet series, DNA weight IUB; Protein gap parameters, residue-specific penalties on, hydrophilic penalties on, hydrophilic residues GPSNDQERK, gap separation distance 4, end gap separation off).
  • the percentage identity is then calculated from the multiple alignment as (N/T)*100, where N is the number of positions at which the two sequences share an identical residue, and T is the total number of positions compared.
  • percentage identity can be calculated as (N/S)*100 where S is the length of the shorter sequence being compared.
  • the amino acid/polypeptide/nucleic acid sequences may be synthesised de novo, or may be native amino acid/polypeptide/nucleic acid sequence, or a derivative thereof.
  • a substantially similar nucleotide sequence will be encoded by a sequence, which hybridises to any of the nucleic acid sequences referred to herein or their complements under stringent conditions.
  • stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 6x sodium chloride/sodium citrate (SSC) at approximately 45 0 C followed by at least one wash in 0.2x SSC/0.1% SDS at approximately 5-65 0 C.
  • a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the peptide sequences according to the present invention.
  • nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
  • Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
  • Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
  • small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
  • Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
  • the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
  • the positively charged (basic) amino acids include lysine, arginine and bistidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Programs that calculate this value for pairs of protein sequences within an alignment include PROTDIST within the PHYLIP phylogeny package (Felsenstein; http://evolution.gs.washington.edu/ phylip.html) using the "Similarity Table” option as the model for amino acid substitution (P). For DNA/RNA, an identical option exists within the DNADIST program of PHYLIP.
  • the protein according to the first or second aspect which comprises the Lad sequence
  • DNA which comprises a lacO sequence
  • the high affinity between the Lad and lacO sequences enables efficient purification of the DNA from a solution of low purity DNA, such as a crude cell lysate. This is particularly advantageous for producing a high purity solution of DNA, which may then be used in gene therapy, or other uses in which high concentration DNA is required.
  • a method of purifying DNA comprising at least a region of lacO operator sequence or variant thereof, the method comprising the steps of:-
  • the method according to the invention takes advantage of the surprisingly strong binding affinity between Lacl and lacO of the Lac operon.
  • the Lac operon has three Lad repressor recognition sites, (i) lacOl, (ii) / ⁇ cO2, and (iii) lacO3.
  • the DNA to be purified comprises at least one of (i) lacOl, (ii) lacO2, and (iii) lacO3 sites, or a derivative or variant thereof.
  • the DNA to be purified comprises at least two sites, and more preferably, at least three of (i) / ⁇ cOl, (ii) lacOl, and (iii) lacOZ sites, or a derivative or variant thereof.
  • the lacOl sequence has a stronger affinity for the Lad protein than for lacOl or lacO3, and therefore enables a high rate of plasmid recovery.
  • the DNA to be purified may comprise a functional fragment of the lacO operator sequence.
  • the lacO operator sequence may comprise a variant or functional region of any of the lacOl, lacO2, or lacO3 Lad repressor recognition sites, which are sufficient for Lad binding.
  • the sequence of locOs harboured on the plasmids, pUC19/ ⁇ c o s as described in Example 1 has a sequence, which is a variant fragment of the wild-type l ⁇ cOl sequence, but which still enables a substantial rate of DNA recovery using the method according to the fifth aspect.
  • use of the l ⁇ cOs variant increases the affinity of the fusion protein-DNA interaction by approximately 10-fold.
  • the target DNA comprises the l ⁇ cOs variant operator sequence.
  • the method according to the fifth aspect may be used to purify any DNA sequence, which comprises at least a part of a l ⁇ cO operator sequence or a variant which is able to bind at least partially to Lad repressor.
  • the DNA to be purified comprising the I ⁇ cO sequence is double stranded, preferably, supercoiled, and preferably, circular.
  • the DNA may be in the form of a plasmid.
  • plasmids which include at least a part of a l ⁇ cO sequence or variant thereof, which would bind to Lad repressor, such as a pUC plasmid, pTriEx vectors, pGEX vectors, pUC 18 and 19, pET vectors employing a T71ac promoter i.e.
  • pETlla-d pET15b, pETl ⁇ b, pET19b, pET21a-d (+), pET24a-d(+), pET22a-d(+), pET26a-d(+), pET25b(+), pET27b(+), pET28a-c(+), pET29a-c(+), pET30a-c(+), pET31b(+), pET32a-c(+), pET33b(+), pET39b(+), pET40b(+), pET41a-c(+).
  • pUC19 which has a lacOZ and a lacOl sequence.
  • plasmid DNA Current technology used to purify plasmid DNA consists of growing a culture of micro-organisms in which the plasmid is harboured to produce sufficient biomass, and then isolating the resultant plasmid DNA.
  • the host may be a eukaryote (e.g. yeast) or prokaryote (e.g E.coli), and normally, an antibiotic is included in the culture to provide positive selection so that the host maintains the plasmid during the growth phase at sufficiently high copy numbers.
  • the cells are recovered by centrifugation, and then disrupted by chemical means, or by sonication, to produce a crude cell lysate, thereby releasing the cell's contents, including the desired plasmid.
  • the plasmid DNA normally has to be further purified by use of commercially available kits (such as Wizard preps from Promega, Qiagen kits, Roche GenePure), or by ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, thiophilic chromatography, gyrolite adsorption, or hydroxyapatite chromatography.
  • commercially available kits such as Wizard preps from Promega, Qiagen kits, Roche GenePure
  • ion exchange chromatography reverse phase chromatography
  • size exclusion chromatography size exclusion chromatography
  • thiophilic chromatography thiophilic chromatography
  • gyrolite adsorption gyrolite adsorption
  • hydroxyapatite chromatography hydroxyapatite chromatography
  • the method according to the fifth aspect of the invention is particularly advantageous as it is a very fast, cheap and effective way of obtaining high purity DNA without the need to perform numerous purification and washing steps or ion exchange.
  • RNaseA is normally sourced from bovine material, and is therefore not a desirable component of the purification method, particularly for DNA, which will have therapeutic uses.
  • yields of the target DNA are increased than that achieved in the presence of RNaseA, as described in Example 2.
  • the DNA being purified according to the present invention may be contained within a solution, for example, a crude cell lysate.
  • a solution for example, a crude cell lysate.
  • the DNA comprises plasmid DNA, which has been made by bacteria, (e.g. E.coli DH5 ⁇ )
  • the DNA may be easily purified from the crude lysate following cell lysis.
  • the crude cell lysate containing the target DNA may require pre-purification by suitable means, for example, by alkaline lysis.
  • the protein according to the first or second aspect of the invention is prepared by expressing the nucleic acid of the third aspect, in a host organism, (e.g. E.coli BL21-Gold), for example, by an expression vector according to the fourth aspect.
  • a host organism e.g. E.coli BL21-Gold
  • the protein according to the first or second aspect may be contained within a solution, for example, a crude cell lysate.
  • the fusion protein and the DNA to be purified may both be prepared by growing up in suitable host cells until sufficient respective concentrations are reached. Suitable methods for determining biomass concentration of the host organisms and therefore the respective concentrations of the target DNA and fusion protein would be known to the skilled technician.
  • the host cells would then be lysed by suitable means, for example, by alkali lysis or sonication, thereby producing two crude cell lysates, one containing the fusion protein and the other containing the target DNA.
  • the presence of the marker protein (GFP) on the fusion protein enables a very quick determination of the concentration of the fusion protein.
  • the two cell lysates may then be simply added together for sufficient time so that the Lacl repressor protein on the fusion protein and the lacO operator sequence or variant thereof, bind together.
  • one cell lysate containing the fusion protein may be added to a solution containing pre-purified target DNA.
  • the conditions required to allow suitable LacI// ⁇ cO binding will be known to those skilled in the art, and will depend on the specific target DNA. A suitable methodology is described in the Examples. The inventors were very surprised that the fusion Lad protein could be contained with a crude cell lysate and still manage to bind with the lacO sequence in the target DNA.
  • the fusion Lad protein may be produced prior to use in the method according to the fifth aspect, as described in Example 2.
  • the crude cell lysate containing the Lad fusion protein may be frozen or lyophilised using known techniques, and then stored at room temperature.
  • the lyophilised lysate may be re-constituted, for example, by adding a suitable buffer, such as PBS.
  • the re-constituted crude extract containing the fusion protein may then be added to the crude extract containing the target DNA.
  • the support surface may be any surface adapted to bind to the protein in accordance with the first or second aspect of the invention, and preferably the linker peptide thereof.
  • the support surface may comprise a resin, which may be porous.
  • the support surface may be in the form of a column or beads. Alternatively, the support surface may be a membrane.
  • suitable support surface ⁇ include functionalised media appropriate to the protein linker (eg. TALONTM resin or HisLinkTM Resin or IMAC tagged magnetic beads such as MegneHisTM beads for the His 6 linker, or Glutathione Sepharose for GST).
  • the support surface is functionalised with a metal ion that is suitably adapted to bind to the protein linker, for example, Co 2+ ions.
  • the linker on the protein comprises a His6 cross-linker
  • the support surface comprises a composition adapted to bind with His6 repeats, such as an IMAC resin.
  • the protein-DNA complex interacts with the IMAC resin by the coordination between His 6 and Co 2+ .
  • Co 2+ is better than between His 6 and Ni 2+ , which is more frequently used with MAC resins.
  • the DNA to be purified is mixed with the protein according to the first or second aspect, so that the DNA/protein complex is allowed to form first, before the DNA/protein complex is added to the support surface.
  • the inventors have noticed that far better DNA purification is achieved by allowing the DNA and protein to complex before adding to the support surface.
  • the eluting step is carried out by washing the support surface to which the protein/DNA complex is attached, with an allosteric inducer of the Lad repressor protein.
  • the inducer molecule binds to the inducer binding domain of the protein according to the first or second aspect. Addition of the inducer causes a conformational change of the Lad protein, thereby resulting in the Lad repressor becoming detached from the lacO sequence on the target DNA being purified. The result of this is that the DNA being purified is released from the fusion protein, and hence, support surface, and is therefore eluted therefrom.
  • the inducer may be IPTG or allolactose. If IPTG is used to elute the target
  • the inducer is used in conjunction with salt, for example, a group I metal chloride, such as sodium chloride.
  • salt for example, a group I metal chloride, such as sodium chloride.
  • a 50OmM concentration solution is preferred.
  • the inducer comprises a solution of IPTG or allolactose, and sodium chloride.
  • the method comprises at least one washing step of recovering unbound material, i.e. DNA and protein which has not formed a DNA/protein complex, and/or which has not successfully bound to the support surface, prior to elution from the support surface.
  • the support surface may be a column or beads, which is agitated to rinse off the non-bound or non-complexed fusion protein.
  • the washing step may comprise rinsing of the column in a suitable buffer, for example, PBS by gravity flow.
  • the method comprises a step of removing as much of the protein bound to the support surface as possible. This is referred to as a stripping step.
  • the stripping may be achieved by adding acid, alkali, or a chelating agent to the support surface.
  • the support surface is then re-functionalised or recharged with the metal ion adapted to bind to the protein linker, for example, Co 2+ ions.
  • the process may be repeated, by adding fresh, crude protein lysate to the support surface.
  • kits comprising a protein according to either the first or second aspect, and a support surface adapted to bind thereto.
  • the kit may be used as follows. First, target DNA comprising lacO sequence is grown up by culture. The cells are then lysed to form a crude cell lysate.
  • the kit may therefore comprise means to lyse the cells containing the target DNA, for example, reagents for alkaline lysis.
  • the cell lysate containing the target DNA is then added to the protein of the first or second aspect, so that the DNA/protein complex is allowed to form first, before the support surface.
  • the kit comprises the fusion protein within an extract of E.coli, which is preferably lyophilised.
  • the support surface of the kit comprises a resin, which may be porous.
  • the support surface may be in the form of a column or beads or a membrane.
  • suitable support surfaces include functionalised media appropriate to the linker peptide in the protein (eg. TALONTM resin or HisLinkTM Resin or EVIAC tagged magnetic beads such as MegneHisTM beads for the His 6 linker, or Glutathione Sepharose for GST).
  • the kit comprises means to separate the support surface, e.g. a column.
  • the kit may further comprise a desalting column.
  • the kit may comprise a wash buffer and, preferably an elution buffer.
  • the kit may further comprise means to strip the support surface of the fusion protein.
  • the kit may comprise means to re-functionalise the support surface, preferably, with Co 2+ ions.
  • Figure 1 shows a schematic representation of Lacl-based affinity capture of plasmid DNA in accordance with an embodiment of the invention
  • Figure 2 shows a fluorescence profile for an increasing concentration (nM) of lacI-His-GFP incubated with 100 nM Streptavidin-Biotin immobilised ds lacOs (circle). Fluorescence is expressed in arbitrary units. The effect of subsequently incubating with 5 mM IPTG for 2 hr is also shown (square).
  • Figure 3 shows electrophoretic gels of Lacl-based affinity capture of pUC19 operator variants.
  • Figure 4 shows purity analysis of the affinity purification fractions.
  • A Silver stained SDS-PAGE analysis of the samples taken at each stage of pUC19/ flC o 3 // ⁇ c os plasmid purification;.
  • B Samples taken at each stage of plasmid purification were analysed for the presence of GFP as described m. ⁇ iotechniques 35, 988-996 (2003)
  • Figure 5 shows affinity purification of plasmid DNA directly from crude lysates
  • Figure 6 shows electrophoretic gels of affinity purification of pUC19/ ⁇ c o 3 // ⁇ C os from crude bacterial lysate with a second crude bacterial lysate containing approximately (A), 4 nmol; (B), 2 nmol; (C), 1.5 nmol; (D), 1 nmol; (E), 750 pmol; (F), 500 pmol; (G), 250 pmol; (H), 100 pmol, (I), 50 pmol; (J), 10 pmol and (K), 1 pmol LacI-His 6 -GFP;
  • Figure 7 shows purity analysis of affinity purification fractions.
  • A purification with - 1.5 nmol LacI-His 6 -GFP;
  • B purification with ⁇ 1 nmol Lacl- HiS 6 -GFP;
  • Figure 8 there is shown an electrophoretic gel of affinity purification of plasmid DNA in the absence of RNaseA treatment.
  • FIG. 9 there is shown affinity purification of plasmid DNA with lyophilised, crude E. coli lysate as the affinity adsorbent at (A), 750 pmol; (B), 500 pmol; and (C), 250 pmol.
  • the inventors wished to investigate whether protein-DNA interaction between the 274 kDa tetrameric Lad repressor fusion protein and the LacO native operator in a 1.8 MDa pUC19 plasmid would be of sufficient strength to allow capture, and hence, purification of such plasmids.
  • the inventors also wanted to examine whether a low affinity LacO operator on its own in a plasmid would be sufficient to allow its capture and purification, or whether high affinity, synthetic operator sequences be required to permit capture of the plasmid by the Lad protein.
  • the inventors investigated the ability of the Lad protein to capture plasmids containing various combinations of lac operators using a unique fusion protein, in accordance with the invention.
  • the fusion protein consists of the Lad repressor protein fused to a poly-histidine (His ⁇ ) cross-linker or tag, itself fused to Green
  • GFP Fluorescent Protein
  • Figure 1 shows a schematic representation of the Lacl-based affinity capture of plasmid DNA in accordance with an embodiment of the invention. Plasmid DNA is bound by the Lad repressor protein tetramer, which is then immobilised via a
  • the GFP in the fusion protein permits localization and estimation of protein concentration.
  • the inventors had determined that the Lad protein retains it's DNA binding affinity for lacO, they then engineered several variants of pUC19, with combinations of both native and synthetic operators as listed in Table 1.
  • Base numbers refer to positions in wild type pUC19, which has native operators lacO3 and lacOl (1 st and 2 nd operators respectively).
  • X ⁇ cOs refers to a synthetic operator, in which, differences between native operators are underlined, with spaces representing deletions.
  • Lac operator mutants were constructed from the following primer/template mixtures: pUC19 / ⁇ cO3 5'-TTT CAC ACA GGA AAC AGC TAT-'3 (SEQ ID No.24) & 5'-TTC CAC ACA ACA TAC GAG CCG-'3 (SEQ ID No.l) (template pUC19, annealing @ 48°C); pUC Im me diate 5'-GGA ATT GTG AGC GGC TCA CAA TTT CAC ACA G-'3 (SEQ ID No.25) & 5'-ACA CAA CAT ACG AGC CGG-'3 (SEQ ID No.2) (template pUC19, annealing @ 53 "C); pUC19 / ⁇ cO3 // ⁇ c os 5'-CTC ACA ATT TCA CAC AGG AAA CAG CTA-'3 (SEQ ID No.26) & 5'-CGC TCA CAA TCC CAC ACA ACA T
  • AU PCRs were performed for 30 cycles of 1 min. @ 95 0 C, 1 min. @ specified annealing temperature, 5 min. 45 sec. @ 72 0 C in the presence of O.lng of template plasmid, 50 pmols each primer " , 100 nM dNTPs, 3 units Pfu DNA polymerase and Ix Pfu buffer, reaction volumes 100 ⁇ l.
  • PCR products were 5' phosphorylated, self-ligated and transformed into E.coli DH5 ⁇ cells. Plasmids were isolated from resulting colonies and sequenced to confirm the presence of required mutations. Oligonucleotides were obtained from MWG-Biotech, Germany. DNA sequencing was performed by the Functional Genomics Lab, University of Birmingham, UK.
  • the LaCl-HiS 6 -GFP fusion protein was then constructed as follows.
  • AAA CAG CTA TGA CCA-3 (SEQ ID No.6) & 5'-TAG TTA TCC TGG CTC ATA TTT CCA CAC AAC ATA CGA GCC GGA AGC-'3 (SEQ ID No.7).
  • PCR was performed as described above, with annealing at 60°C and an extension time of 7 min.
  • the product was cloned as described above and DNA isolated using a Wizard- plus Miniprep kit (Promega).
  • the Lad gene was amplified from vector pLacI (Novogen, UK), with primers 5'-GGG AAG CTT GGT GAA ACC AGT AAC GTT A-'3 (SEQ ID No.8) & 5'-AAA CTC TAG AGT CTG CCC GCT TTC CAG-'3 (SEQ ID No.9), which inserted Hindl ⁇ l and Xba ⁇ restriction sites at the 5' and 3' ends of the amplicon, respectively.
  • PCR conditions were as above with annealing at 53 °C and an extension time of 2.5 min.
  • the PCR product was restricted with HmdIII and Xbal for 3 hr at 37 0 C and ligated into similarly digested pGFPuv that had been modified to remove the lacO element as described above.
  • the resulting LacI-GFP vector was cloned and sequenced.
  • a His 6 cassette was prepared by hybridising 1 nmol of oligonucleotides 5'-CTA GAG CAC CAT CAC CAT CAC CAT CGG GTA C-'3 (SEQ ID No.10) & 3'-TC GTG GTA GTG GTA GTG GTA GCC-'5 (SEQ ID No.11) (previously 5' phosphorylated with T4 polynucleotide kinase) in a 50 ⁇ l reaction containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl 2 , 4 % (w/v) PEG 8000.
  • the mixture was heated to 95 °C for 4 min., followed by progressive cooling to 25 0 C at a rate of 1 °C per min and the resulting cassette was ligated into the LacI-GFP vector that had been restricted with Xbal, Kp ⁇ l and dephosphorylated with CIP (New England Biolabs, UK).
  • the resulting construct was cloned as described above and sequenced.
  • the LacI-His 6 -GFP construct was transformed into E. coli BL21-Gold cells (Stratagene) and the encoded protein expressed constitutively.
  • a single fresh colony was inoculated into 200 ml of LB media supplemented with 50 ⁇ g per ml ampicillin in a 1 litre flask and incubated with shaking at 30 0 C, overnight.
  • This culture (1 ml) was used to inoculate a 10 1 BioFlow 3000 fermentor (New Brunswick) containing the same medium and the resulting culture was fermented overnight at 250 rpm, pH 7.0, 30 °C with O 2 saturation.
  • the cells were then recovered by centrifugation at 4000 g, and the pellets resuspended in an equivalent culture volume of PBS (138 mM NaCl,
  • the pellets were then re-suspended in PBS (pH 7.4) at a 10-fold concentration of the original culture volume (25 ml) and 1 ml fractions aliquotted, centrifuged and stored dry at -80°C. Aliquots were resuspended as required in 1ml PBS, lysed by sonication (Misonix microson ultrasonic cell disrupter; 4 x 3 s sonication at maximum power, followed by incubation on ice for 2 min, 4 repeats). The resulting lysate was clarified by centrifugation at 21,000 g for 10 min. at 4 0 C. The sonication and centrifugation was then repeated and the supernatant recovered.
  • PBS pH 7.4
  • LacI-His-GFP protein The concentration of LacI-His-GFP protein was estimated by measuring the GFP fluorescence using the method described in Biotechniques 35, 988-996 (2003). Crude cell lysate was stored at 4 °C and used the same day. Protein yield was typically between 5 - 15 mg of soluble LacI-His-GFP per 1 of culture.
  • DNA in a total volume of 10 ml PBS (pH 7.4) and mixed on a flatbed roller for 2 hr at room temperature. Meanwhile, 3ml of pre-equilibrated TALONTM resin (BD Biosciences, prepared according to the manufactures instructions) was placed in a 15 ml sintered disposable polypropylene column and drained.
  • pre-equilibrated TALONTM resin BD Biosciences, prepared according to the manufactures instructions
  • the protein-DNA mixture was added, the column capped and incubated for 2 hr. on a flatbed roller at room temperature. Unbound material was then removed by gravity-flow and the resin was washed with 3 x 10 ml PBS.
  • Plasmid DNA was eluted with 2 x 3 ml elution buffer (10 mM Tris-HCl (pH 7.4), 1 mM IPTG, 500 mM NaCl) and the eluates desalted by passing through a PD-10 desalting column (Amersham Pharmacia Biotech) according to manufacturer's instructions.
  • the NaCl/EPTG solution induced an allosteric change in Lad, so allowing release of the plasmid, whilst the protein ligand remains bound to the resin.
  • Eluted plasmid was desalted and analysed by 0.7 % agarose electrophoresis, 10 % SDS-PAGE and for GFP fluorescence as described in Biotechniques 35, 988-996 (2003). Samples from throughout the process were quantified for DNA, as summarised in Table 2.
  • Lanes: M Molecular Weight marker (420 ng); 1, crude E.coli lysate (equivalent to protein in 15 ⁇ l of binding reaction); 2, complex of plasmid DNA and crude E.coli lysate prior to immobilization; 3, unbound material after immobilization; 4-6, washes 1-3 respectively; 7, elution 1; 8, elution 2; P, purified plasmid DNA ( ⁇ 500 ng). Unless otherwise stated, all lanes correspond to a 15 ⁇ l aliquot of the relevant sample.
  • FIG 4 which shows purity analysis of the affinity purification fractions.
  • (/4) Silver stained SDS-P AGE analysis of the samples taken at each stage of pUC19i ac o3/i ac os plasmid purification. Lanes: M, Molecular Weight marker (420 ng); 1, complex of plasmid DNA and crude E.coli lysate prior to immobilization; 2, unbound material after immobilization; 3-5, washes 1-3 respectively; 6, elution 1; 7, elution 2. Unless otherwise stated, all lanes correspond to a 15 ⁇ l aliquot of the relevant sample(5). The samples taken at each stage of plasmid purification were analysed for the presence of GFP as described previously. Immobilized values were calculated from the protein/DNA mix and unbound fractions. AU samples correspond to 100 ⁇ l aliquots (averaged triplicates) of the relevant fraction. .
  • the affinity between Lad and the operator sequence correlates with the degree of plasmid capture.
  • the greatest immobilization is mediated by dual lacO3 I lacOs operators on the pUC19 / ⁇ c o 3// ⁇ C ⁇ s plasmid.
  • Excellent immobilisation is mediated by wild type lacO3 and variant lacOl operators on the pUC19w ⁇ plasmid, and also by the variant lacOl operator on the pUC19/ ⁇ c o s plasmid.
  • plasmid capture is also possible with a single, low affinity operator
  • Plasmid DNA release is effected by an allosteric mechanism. Allolactose and its analogues induce a conformational change in the Lad structure, rendering it incapable of binding to the operator sequence. Under such circumstances, it is unlikely that the affinity between protein and DNA would affect the efficiency of DNA release.
  • the high molecular weight band in the elution of plasmid DNA (lanes 7-8, Fig 3 a) is consistent with the silver staining of plasmid DNA itself (lane 9, Fig 3a).
  • Silver staining typically detects as little as 0.25 ng of protein, whereas a BCA assay may detect > 5 ⁇ g of protein.
  • the inventors have determined previously that the minimal protein concentration detectable by the fluorescence assay is 0.4 nM GFP.
  • the resultant solution did not undergo purification by ion exchange chromatography as in section (3) above).
  • the pH of the supernatant was adjusted to 7.0 with 2.5 M NaOH.
  • the supernatant was combined with E.coli BL21-Gold cell lysate containing approximately 2 nmol LacI-His 6 -GFP (as estimated by GFP fluorescence) in a total volume of 50 ml PBS (pH 7.4).
  • the resulting mixture was incubated on a flatbed roller for 2 hr at room temperature. Meanwhile 4 ml of TALONTM resin was prepared as described above.
  • the affinity isolation process was then performed as described above. Plasmid DNA was eluted with 5 ml elution buffer and the eluate desalted. AU fractions were analysed as described above.
  • HiS 6 -GFP protein was combined with crude E.coli lysate containing pUC19 / ⁇ c o 3// ⁇ c ⁇ s plasmid DNA, in a 50 ml final volume.
  • the complex was immobilized on IMAC resin, washed (3 x 10 ml PBS pH 7.4) and plasmid DNA eluted (I x 5ml elution buffer) and desalted.
  • LacI-His 6 -GFP protein (equivalent to protein in 15 ⁇ l of binding reaction); 3, complex of crude E.coli lysates containing plasmid DNA and protein prior to immobilization;
  • the inventors have demonstrated a high-yielding generic, affinity-based plasmid purification process using the LacI-His6-GFP fusion protein in accordance with the invention.
  • the presence of the GFP domain in the fusion protein enables sensitive detection of potential protein leeching, whilst the His 6 tag enables independent release of the plasmid from the adsorbent, without concomitant protein release.
  • the process also works well (81% recovery) with a plasmid containing a single engineered operator sequence, lacOs.
  • Lad protein binds to a combination of the lacOl, lacOl and lacO3 operator sequences and many engineered plasmids, such as pUC19, contain lacOl and lacO3.
  • the interaction between protein and plasmid is stronger when two, rather than one, operator sequences are present.
  • the method in accordance with the invention is very simple and efficient. Crude bacterial lysate containing a Lacl-His ⁇ -GFP fusion protein is first mixed with plasmid that contains lac operator(s) and the resulting protein/DNA complex is isolated utilizing an IMAC resin, which is specific for the His 6 Tag. Plasmid DNA is then eluted with a NaCl/EPTG solution, and desalted. The process is exceptionally specific for plasmid DNA, excluding genomic DNA, RNA and host cell protein, whilst leaching of the affinity adsorbent is also undetectable. The process is even effective as a dual crude lysate approach in which crude cell lysate (protein) is combined with crude, neutralised alkaline lysate (plasmid) to yield apparently pure plasmid DNA, although yields are currently low.
  • the high recovery rates mean that it should be possible to generate milligram quantities of purified plasmid, with approximately 100 ml of resin and 2.5 mg of tetrameric fusion protein.
  • the IMAC resin used in the present experiments is more typically employed in protein purifications and is therefore of high porosity.
  • the porous nature of the TALONTM resin means that the protein-DNA complex is likely to be localized to the periphery of the beads, since the complex is liable to be too large to penetrate deep into their matricies. Indeed, fluorescence microscopy of the immobilised complex suggested that it was almost entirely localised to the periphery of the TALONTM beads (data not shown).
  • plasmid based vectors are biological products, their manufacture is strictly regulated by the FDA and the European Medicines Evaluation Agency (EMEA), whose guidelines indicate that contaminating protein levels must be less than 10 ng per dose of plasmid as measured by silver stained SDS-PAGE and undetectable by BCA assay. Genomic DNA and RNA must also be absent as visualized by agarose gel electrophoresis. Examination of Figures 3 and 4 suggest that the affinity process described herein will fall within these guidelines. In conclusion, it is now possible to envisage large-scale manufacturing of therapeutic grade plasmid DNA utilizing affinity based processes that are synonymous with protein purification, but which have previously eluded DNA manufacture.
  • EMEA European Medicines Evaluation Agency
  • Example 1 demonstrated that the fusion protein ligand, LacI-His 6 -GFP, could be employed in a dual crude lysate approach, whereby one crude bacterial lysate is required to supply the plasmid DNA and another it required to supply the protein- based affinity ligand. As discussed in Example 1 , this procedure generated remarkably pure plasmid DNA. The inventors then decided to investigate whether it would be possible to use the fusion protein according to the invention, and optimise the "dual crude lysate" procedure to produce high process yields of target DNA.
  • the cells were then re-suspended in 12 ml of 50 mM Tris-HCl (pH 7.4) with or without 100 ⁇ g ml "1 RNaseA and were subsequently alkaline lysed using a Roche Genopure Kit according to manufacturer's instructions. Following flocculent removal, the pH of the supernatant was adjusted to 7.4 with approximately 4.5 g of Tris base (Sigma).
  • E. coli BL21-Gold cell lysate prepared as described in Example 1 containing an appropriate concentration of Lacl- HiS 6 -GFP as indicated in individual experiment, where LacI-His 6 -GFP concentration was estimated by GFP fluorescence as described in Biotechniques 35, 988-996.
  • the resulting mixture was incubated for two hours in a 50 ml FalconTM tube on a flatbed roller at room temperature. Meanwhile, 5ml TALONTM resin suspension was equilibrated with PBS (pH 7.4) according to the manufacturer's instructions. The resin was then mixed with the protein-DNA mixture and incubated for two hours on a flatbed roller at room temperature.
  • the resin was subsequently recovered using a sintered polypropylene column and the flow through collected by gravity flow.
  • the resin was washed with 3 x 10 ml
  • the concentration of the affinity ligand (the fusion protein) can be estimated within a bacterial lysate by measuring GFP fluorescence, direct quantitation of plasmid DNA within crude lysates is problematic. Therefore, to optimise subsequent plasmid recovery, a range of quantities of the protein lysate was employed to establish the best complexation ratio.
  • agarose gel electrophoresis of the collected fractions (as shown in Figure 6 (A-K)) demonstrates that plasmid DNA can indeed be affinity captured directly by combination of the two bacterial crude lysates. Furthermore, it can be seen that only one species of DNA (supercoiled) is purified (lane 8), and that no visible genomic DNA or residual RNA is recovered. Picogreen quantitation of the eluted plasmid DNA was then used to estimate the concentration of eluted plasmid DNA, as follows. A stock solution of Picogreen in DMSO (Molecular Probes) was stored in the dark at -20 0 C and diluted 200-fold in PBS to working concentration, when required.
  • Picogreen quantitation of the eluted plasmid DNA was then used to estimate the concentration of eluted plasmid DNA, as follows. A stock solution of Picogreen in DMSO (Molecular Probes) was stored in the dark at -20 0 C and diluted 200-fold in PBS to working concentration, when required
  • De-salted plasmid eluate was diluted 25, 50, and 100-fold in PBS and 100 ⁇ l aliquots mixed with 100 ⁇ l aliquots of diluted Picogreen solution in a Nunc MaxiSorp 96-well plate. The plate was then incubated at room temperature with gentle agitation for 5 minutes. After incubation the fluorescence was recorded on a SpectraMax Gemini XS plate reader (Molecular Devices) with an excitation and emission wavelength of 480 nm and 520 nm respectively with a cut-off of 515 nm. All measurements were performed in triplicate. The Picogreen fluorescence was then measured against a standard curve prepared with pUC19 DNA (MBI Fermentas) and the concentration of eluted DNA calculated.
  • MBI Fermentas pUC19 DNA
  • DNA recovery is achieved when approximately 250 pmols of crude protein lysate is mixed with the neutralised alkaline lysis supernatant from 200 ml of E. coli culture, as shown in Table 3.
  • Figure 7 demonstrates that no host protein is co-purified with the plasmid DNA.
  • the high molecular weight band visualised in the eluate is the plasmid DNA itself.
  • the volume of required solid support was then investigated.
  • the quantity of resin employed was both halved and doubled. Examination of the collected fractions demonstrated that halving the resin also halved plasmid yield, whilst doubling the quantity of resin increased plasmid yield, but not in proportion to the increase in resin volume (data not shown).
  • the inventors therefore selected experimental conditions of 250 pmol affinity ligand : plasmid DNA from 200 ml E. coli culture, purified over 5ml of resin for future experiments.
  • FIG. 8 there is shown affinity purification of plasmid DNA in the absence of RNaseA treatment.
  • E. coli (200 ml) containing the pUC19/ ac o 3 // ⁇ C ⁇ s plasmid was harvested by centrifugation, re-suspended in 50 mM Tris-HCl pH 7.4 and subjected to alkaline lysis. After adjustment to pH 7.4, the affinity purification procedure was performed as previously described, with crude E. coli lysate containing approximately 250 pmol of LacI-His 6 -GFP. Lanes: M, molecular weight marker (420 ng); 1, crude E.
  • RNaseA (as illustrated Table 3).
  • E. coli BL21-Gold cell lysate containing the LacI-His 6 -GFP fusion protein was prepared, and the LacI-His 6 -GFP concentration estimated, as described previously in Example 1.
  • the lysate (containing 250, 500, and 750 pmol of LacI-His 6 -GFP fusion protein) was subsequently frozen in 5 ml bijou tubes by immersion in liquid nitrogen for 5 minutes and then immediately freeze dried under vacuum for a period of approximately 24 hours using a Savant MicroModulyo. Lyophilised protein samples were stored at room temperature for two weeks until required and then re-constituted with 2 ml PBS. Subsequent crude lysate plasmid purification was carried out as stated above. The lyophilised crude protein lysate was then re-constituted and the plasmid purification experiments repeated, as illustrated in Figure 9.
  • FIG. 9 there is shown affinity purification of plasmid DNA with lyophilised, crude E. coli lysate as the affinity adsorbent.
  • the lysates were re- . constituted in 2 ml PBS pH 7.4 and each was combined crude plasmid lysate from a 200 ml E. coli culture (no RNaseA treatment).
  • Affinity purification was performed as previously described.
  • B Affinity purification with ⁇ 500 pmol LacI-His 6 -GFP
  • C Affinity purification with ⁇ 250 pmol LacI-His 6 -GFP.
  • Plasmid yield was comparable (cf. Tables 3 and 4) irrespective of whether the affinity lysate was prepared fresh or lyophilised and stored at room temperature. Moreover, PCR-based analysis of eluted plasmid detected no contamination by E. coli genomic DNA.
  • plasmid DNA can be purified directly from a bacterial crude lysate utilising a Lad fusion protein (itself part of a crude bacterial lysate) as an affinity ligand, without the need for RNaseA treatment. Even using current batch processes, the inventors believe that their yields are scalable for bio-manufacturing.
  • the affinity ligand may be prepared in advance (in large quantity), lyophilised and stored at room temperature for use when required. Thus, a 10 1 fermentation would provide sufficient affinity ligand to purify 50 mg of plasmid DNA.
  • process yields are likely to be improved even further by the use of a non-porous, micropellicular support (J. Chromatogr., 806, 3-30), a niacroporous monolith ⁇ J.Sep.Sci, 27, 819-827), or adsorptive membranes (J. Chromatogr A., 989, 165-173) as opposed to the current porous MAC resin.
  • a non-porous, micropellicular support J. Chromatogr., 806, 3-30
  • adsorptive membranes J. Chromatogr A., 989, 165-173
  • DNA-binding proteins are not generally as robust as antibodies and are unlikely to be stable to acid washes etc. Therefore, because the inventors do not anticipate re ⁇ using the ligand that is immobilised on the resin, they used a crude protein lysate, rather than a purified affinity ligand. Hence, the advised strategy would be to strip the resin completely after plasmid elution (by acid or chelating agents for example), recharge the column with the metal ion (Co 2+ ) and repeat the process with fresh, crude protein lysate. Clearly this would be prohibitively expensive if the protein were itself purified first, but is both trivial and cheap if the protein is supplied as a simple, lyophilised crude lysate. As discussed previously in Example 1, plasmid DNA should not be eluted with IPTG if affinity purification is employed as a downstream process. Rather, a safer, non-toxic alternative such as allolactose should be used to prevent potential toxicity concerns.
  • Example 1 using conventionally purified plasmid DNA and a crude bacterial lysate to supply the protein ligand, low levels of E. coli genomic DNA were detected by PCR in eluted plasmids. However, similar analysis of the plasmid
  • Lad repressor Protein Sequence (SEQ ID No. 12) MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAELNYIPNRVAQQLAGKQS LLIGVATSSLALHAPSQ ⁇ VAAIKSRADQLGASVWSMVERSGVEACKTAVHNLLAQRVSGLIIN YPLDDQDAIAVEAACTNVP ALFLDVSDQTPINSIIFSHEDGTRLGVEHLV ALGHQQIALLAGPL SSVSARLRLAGWHKYLTRNQIQPIAEREGDWSAMSGFQQTMQMLNEGIVPTAMLVANDQMA LGAMRAITESGLRVGADISVVGYDDTEDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVK GNQLLPVSLVKRKTTLAPNTQTASPRALADSLMQLARQVSRLESGQ
  • MKPVTLYDVAEYAGVSYQTVSRWNQASHVSAKTREKVEAAMAEL Domain Residues 46 - 62 of SEQ ID No. 12 (SEQ ID No. 15)

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Abstract

L'invention porte: selon un premier aspect sur une protéine comprenant le peptide répresseur LacI et une séquence du peptide lieur His3-20, et selon un deuxième aspect sur une protéine comprenant le peptide répresseur LacI comprenant une séquence d'acide aminé correspondant sensiblement à la séquence SEQ ID No. 12 de la description ou l'une de ses variantes fonctionnelles, et un peptide lieur pouvant se fixer à une surface support.
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GOEL APOLLINA ET AL: "Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1523, no. 1, 1 September 2000 (2000-09-01), pages 13 - 20, XP002336173, ISSN: 0006-3002 *
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