WO2011072479A1 - Protéine de fusion contenant une protéine de liaison à l'adn simple brin et procédés d'expression et de purification associés - Google Patents

Protéine de fusion contenant une protéine de liaison à l'adn simple brin et procédés d'expression et de purification associés Download PDF

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WO2011072479A1
WO2011072479A1 PCT/CN2010/002000 CN2010002000W WO2011072479A1 WO 2011072479 A1 WO2011072479 A1 WO 2011072479A1 CN 2010002000 W CN2010002000 W CN 2010002000W WO 2011072479 A1 WO2011072479 A1 WO 2011072479A1
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pssb
seq
protein
stranded dna
fusion protein
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PCT/CN2010/002000
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English (en)
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Daochun Kong
Yu Hua
Jiazhi Hu
Jingya Sun
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Daochun Kong
Yu Hua
Jiazhi Hu
Jingya Sun
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Publication of WO2011072479A1 publication Critical patent/WO2011072479A1/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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • the present invention generally relates to the technologies of protein expression and purification, and more particularly to a fusion protein containing a single- stranded DNA binding protein and methods for expression and purification of the fusion proteins containing a single-stranded DNA binding protein.
  • a number of approaches have been developed for the isolation and purification of proteins, particularly recombinant proteins, from other components of a biological sample.
  • the approaches include ion exchange chromatography based on molecular charges, gel filtration based on molecular size, and affinity chromatography.
  • the affinity chromatography is more specific and much more efficient than the other purification approaches because it makes use of the specific affinity of a protein for a purifying reagent such as an antibody or ligand to which it specifically binds.
  • One member of a binding pair may be used to "tag" a protein of interest, with the other member used as an affinity ligand.
  • Such a protein "tag” may be "fused” recombinantly and expressed to produce a fusion protein with the tag attached.
  • the "tagged" fusion protein is then affinity purified by interaction with the binding partner of the tag and the tag is then optionally cleaved to release pure protein.
  • affinity chromatography suffers from the drawbacks such as unsatisfactory purity, time-consuming, high cost and/or unusual elution conditions.
  • One aspect of the present invention provides an expression vector comprising a promoter and a polynucleotide sequence encoding a fusion protein; wherein the fusion protein-encoding polynucleotide sequence is so operably linked to the promoter that the transcription of the fusion protein-encoding polynucleotide sequence is controlled by the promoter; wherein the fusion protein comprises a single-stranded DNA-binding protein and an interest protein or polypeptide fused directly or indirectly with the COQH- terminus or NH 2 -terminus of the single-stranded DNA-binding protein; and wherein the fusion protein is capable of binding to single-stranded DNA.
  • the method comprises contacting a host cell with an expression vector, wherein the expression vector comprises a promoter and a polynucleotide sequence operably linked to the promoter, wherein the polynucleotide sequence encodes a fusion protein comprising a single-stranded DNA binding protein and the interest protein fused directly or indirectly with the COOH-terminus or NH 2 -terminus of the single-stranded DNA binding protein, and wherein the fusion protein is capable of binding to single-stranded DNA; culturing the host cell under such conditions that the fusion protein is expressed; lysing the host cell to obtain a cell lysate; contacting the cell lysate with a substrate immobilized with single-stranded DNA to allow the fusion protein to bind to the single-stranded DNA of the substrate; washing the substrate to remove impurities; and eluting the bound fusion protein from the substrate;
  • Another aspect of the present invention provides a fusion protein comprising a single-stranded DNA binding protein and an interest protein or polypeptide fused directly or indirectly with the COOH-terminus or NH 2 -terminus of the single- stranded DNA binding protein, wherein said fusion protein is capable of binding to single- stranded DNA; thereby the fusion protein can be purified by a substrate immobilized with single-stranded DNA.
  • the fusion protein is soluble and can be purified from cell lysates under physiological condition by affinity chromatography on a column of single-stranded DNA (ssDNA) cellulose.
  • the separation of the said fusion protein from other proteins and impurities existed in cell lysates through one step of ssDNA-cellulose chromatography is highly efficient, apparently more efficient than the other affinity chromatography such as Ni 2+ -agarose column.
  • the fusion protein can be eluted from ssDNA-cellulose by just raising salt (NaCl or KC1) concentration; The recovery of the said fusion proteins from ssDNA-cellulose is very efficient and simple.
  • FIG 1A shows the structure of the series of expression plasmid vectors of pSSB-B, including the schematic representation of pSSB-Bl .
  • This series of plasmids are applicable in bacteria.
  • An additional 6His-tag is located at the N-terminus of the SSB in pSSB-B3 and pSSB-B4 vectors.
  • the multiple enzymatic linkers in pSSB-Bl, pSSB-B2, pSSB-B3 and pSSB-B4 are listed as SEQ ID NOs 3, 4, 5, or 6 respectively.
  • FIG IB shows the structure of the series of expression vectors of pSSB-Y, including the schematic representation of pSSB-YL
  • This series of plasmids are applicable in the fission yeast S. pombe.
  • the positions of 6 His tag, SSB, enterokinase or thrombin cleavage site, and multiple cloning sites are also indicated in a portion of pSSB-Yl or pSSB-Y2 expression vector.
  • the multiple enzymatic linkers in pSSB-Yl and pSSB-Y2 are listed as SEQ ID NOs 7 or 8 respectively.
  • FIG 1C shows the structure of the series of expression vectors of pSSB-I, including the schematic representation of pSSB-Il. This series of plasmids are applicable in insect cells. Also shown are the specific protease cleavage sites among the vectors of pSSB-Il, pSSB-12, pSSB-I3, pSSB-I4. An additional 6His-tag is located at the N-terminus of the SSB in pSSB-I3 and pSSB-I4 vectors. The multiple enzymatic linkers in pSSB-Il, pSSB-12, pSSB-I3 and pSSB-I4 are listed as SEQ ID NOs 9, 10, 11 or 12 respectively.
  • FIG ID shows the structure of the series of expression vectors of pSSB-H, including the schematic representation of pSSB-Hl. This series of plasmids are applicable in human cells. Also shown are the specific protease cleavage sites among the vectors of pSSB-Hl, pSSB-H2, pSSB-H3, and pSSB-H4. An additional 6His-tag is located at the N- terminus of the SSB in pSSB-H3 and pSSB-H4 vectors. The multiple enzymatic linkers in pSSB-Hl, pSSB-H2, pSSB-H3 and pSSB-H4 are listed as SEQ ID NOs 13, 14, 15 or 16 respectively.
  • FIG 2 shows the expression level of the fusion protein 6His-SSB-Sapl
  • FIG 3A shows the purification of the fusion protein 6His-SSB-Sapl through ssDNA-cellulose column.
  • the total cell extracts were applied to the ssDNA- cellulose column.
  • the unbound proteins and other impurities went through the column.
  • the bound 6His-S SB-Sap 1 was eluted from ssDNA-cellulose at -0.4 M KCl.
  • FIG 3B shows that 6His-SSB-Sapl that was already purified by ssDNA- cellulose chromatography bound to Ni 2+ -agarose well and was eluted at 250 mM imidazole. This result indicates that 6His-SSB-Sapl ca be further purified with Ni 2+ - agarose column if it is needed.
  • FIG 4 shows the purification of the fusion protein S SB-Fen 1 that was in cell lysate through ssDNA-cellulose chromatography.
  • the Fenl is human flap endonuclease 1.
  • the fusion protein SSB-Fenl was overexpressed in E. coli cells.
  • the cell extract containing the SSB-Fenl was applied to ssDNA-cellulose column.
  • the bound SSB-Fenl was eluted at -0.4 M KCl.
  • FIG 5 shows the purification of 6His-GST-Sapl from cell lysate through glutathione-sepharose 4B.
  • the fusion protein 6His-GST-Sapl was overexpressed in E. coli cells.
  • the cell extract containing the 6His-GST-Sapl was applied to Glutathione Sepharose 4B column. After the column was thoroughly washed with a buffer without glutathione, the bound 6His-GST-Sapl was first eluted with 10 mM glutathione. The 6His-GST-Sapl that remained binding to the column of Glutathione Sepharose 4B was further eluted by 0.5% SDS.
  • FIG 6 shows the purification of 6His-Sapl from cell lysate through Ni 2+ -
  • NTA agarose column 6His-Sapl was overexpressed in E. coli cells.
  • the cell extract containing 6His-Sapl was applied to Ni 2+ -NTA agarose column and the column was washed with several column volumes of buffer containing 20 mM imidazole to remove unbound proteins and other impurities.
  • the bound 6His-Sapl was eluted at 250 mM imidazole.
  • FIG 7. shows the purification of SSB-Sapl from human cell lysate.
  • the human cell extract containing SSB-Sapl was applied to ssDNA-cellulose column. By step elution, the majority of the bound SSB- Sapl was eluted at salt concentration of 0.2 to 1.0 M KC1.
  • the SSB-Sapl lane in FIG 7-9 indicates the position of this protein in SDS-PAGE gel.
  • FIG 8 shows the purification of SSB-Sapl from insect cell lysate.
  • SSB- Sapl was overexpressed in insect cells.
  • the insect cell extract containing SSB-Sapl was applied to ssDNA-cellulose column.
  • the majority of the bound SSB-Sapl was eluted at salt concentration of 0.2 to 1.0 M RC1.
  • FIG 9 shows the purification of SSB-Sapl from yeast cell extract.
  • Sapl was overexpressed in the fission yeast S. pombe cells. : The yeast cell extract containing SSB-Sapl was applied to ssDNA-cellulose column. The majority of the bound SSB-Sapl was eluted at salt concentration of 0.2 to 1.0 M KC1.
  • FIG 10 shows the cleavage of 6His-SSB-Sapl by thrombin and the removal of 6His-SSB by Ni 2+ -NTA a sarose column.
  • fusion protein is a chimeric molecule in which the constituent molecules are all polypeptides and are attached (fused) to each other such that the chimeric molecule forms a continuous single chain.
  • the various constituents can be directly attached to each other or can be coupled through one or more peptide linkers.
  • a "linker” as used in reference to a chimeric molecule refers to any molecule that links or j oins the constituent molecules of the chimeric molecule. Where the chimeric molecule is a fusion protein, the linker may be a peptide that joins the proteins comprising a fusion protein.
  • the fusion proteins comprise a single-stranded DNA binding protein (SSB).
  • SSB single-stranded DNA binding protein
  • the SSB used herein refers to any protein or polypeptide that is capable of binding to single-stranded DNA; thus when an interest protein or polypeptide is fused to the C- terminal or N-terrmnal of the SSB, it can be purified by the binding of SSB to the single- stranded DNA.
  • an effective affinity chromatography system for purification of S SB-containing fusion proteins can be established; thereby the interest proteins can be easily expressed and purified.
  • the SSB suitable for the present invention may be derived from natural
  • bacteriophage T7 expresses a single-stranded DNA binding protein (SEQ ID NOs 1 and 2) with a molecular weight of 25.6 kDa (Dunn JJ, Studier FW (1983) Complete nucleotide sequence of bacteriophage T7 DNA and the location of T7 genetic elements. JMol Biol. 166(4):477-535). In a physiological condition, it exists as a homo-dimer and is a very soluble protein. It specifically binds to ssDNA with high affinity and without sequence preference. At 1.0 M of salt concentration, T7 SSB dissociates from ssDNA.
  • the SSBs used in the fusion proteins may be a fragment, mutated form, or a variant of the natural ones.
  • the suitable SSBs may be selected from artificial sequence libraries such as phage display by assaying their capabilities of binding to single-stranded DNAs.
  • the selected sequences may be advantageous over the natural ones when the selected sequences are smaller so that there is no need to remove the SSB from the fusion protein. In many cases, there may be no need to remove the SSB if the SSB-tagged fusion protein has normal biological activities and functions.
  • the fusion protein comprises a cleavable linker that is disposed between the SSB and interest protein, affording the removal of the SSB from the fusion protein by chemical or enzymatic treatment of the fusion protein. It is apparent that the cleavable linker can be disposed at any site of the fusion protein according to a user's desire.
  • the cleavable linker comprises a DNA sequence which codes for an amino acid or a sequence of amino acids which can be cleaved chemically or enzymatically at its C-terminal.
  • Examples of chemical agents useful for cleaving proteins are cyanogen bromide, 2-(2-nitrophenylsulfenyl)-3-bromo-3'-methylindolinium (BNPS-skatole), hydroxylamine, and the like. Cyanogen bromide cleaves proteins at the C-terminal of a methionine residue. BNPS-skatole cleaves at the C-terminal of a tryptophan residue. Hydroxylamine cleaves at the C-terminal of the moiety -Asn-Z- in which Z is Gly, Leu, or Ala. ⁇ .
  • Examples of enzymatic agents useful for cleavage are trypsin, papain, pepsin, plasmin, thrombin, enterokinase, and the like. Each effects cleavage at a particular amino acid sequence which it recognizes. Enterokinase, for example, recognizes the amino acid sequence -(Asp)n-Lys- in which n is an integer from 2 to 4.
  • the fusion protein comprises one or more other purification tags.
  • six histidine residues are fused to the SSB at its N- or C ⁇ - terminals, allowing purification of the SSB-containing fusion protein by a combination of Ni 2+ column and single-stranded DNA-immobilized substrate. After the purification, the portion of SSB and six histidine residues can be removed by chemical or enzymatic cleavage.
  • any known purification tag is suitable here including myc tag, HA tag, Flag-peptide, T3 epitope, alpha-tubulin epitope, T7 gene 10 protein peptide tag, glutathione-S-transferase (GST), strep-tag, bovine pancreatic trypsin inhibitor (BPTI), and maltose binding protein (MBP).
  • the expression vectors are nucleic acid constructs, generated recombinantly or synthetically, with nucleic acid elements that are capable of effecting expression of a gene or cDNA in hosts compatible with such sequences.
  • the recombinant expression vector includes one or more regulatory sequences operably linked to the nucleic acid encoding the enzyme(s) in a manner that allows for transcription of the nucleic acid into mR A and translation of the mRNA into the subject proteins.
  • the term "regulatory sequences" is art-recognized and intended to include promoters, and/or enhancers and/or other expression control elements (e.g. , pdlyadenylation signals). Such regulatory sequences are known to those skilled in the art (see, e.g., Goeddel (1990) Gene Expression Technology: Meth. Enzymol. 185, Academic Press, San Diego, Calif.).
  • the expression vectors are a series of plasmid expression vectors (pSSB) that have been constructed to guarantee SSB-tagged fusion proteins to be expressed in a wide range of host cells: pSSB-Bl - B4 for bacteria, pSSB- Yl - Y2 for yeast, pSSB-Il - 14 for insect cells, and pSSB-Hl - H4 for mammalian cells (FIG 1).
  • a typical expression vector before incorporating a DNA sequence encoding for an interest protein contains: 1) a promoter region, 2) a 5' untranslated region, 3) a protein- coding region, 4) a 3' untranslated region, and 5) a transcription termination site.
  • the "protein-coding region” is so constructed that interest protein sequence can be easily inserted to form a fusion protein.
  • the "protein-coding region” includes a 696bp DNA fragment of Bacteriophage T7 gp2.5 gene encoding for SSB (SEQ ID NO 1).
  • a specific protease (thrombin or enterokinse) recognition linker is inserted near the C-terminal of SSB.
  • Multiple cloning sites are also designed downstream the protease cleavage linker, wherein DNA sequences coding for the desired proteins or polypeptides can be inserted.
  • other purification tags can also be included in the "protein-coding region”. It will be appreciated that desired polypeptides can be operably linked to constitutive promoters or inducible and/or tissue-specific promoters.
  • the expression vectors can be derived from cosmids or viruses.
  • cosmids or viruses For example, replication defective retroviruses, adenoviruses and adeno-
  • associated viruses can be used
  • a yeast artificial chromosome which contains both a centromere and two telomeres, allowing YACs to replicate as small linear chromosomes.
  • YAC yeast artificial chromosome
  • suitable expression systems include, but are not limited to, baculo virus expression vectors.
  • Another aspect of the present invention provides a method for purification of an interest protein in the form of a SSB-tagged fusion protein. Due to the nature that DNA exists at a double-stranded state inside cells, there are a lot of proteins able to bind to dsDNA with various degree of affinity while few proteins have the characteristic to bind to ssDNA; SSB is evolutionary selected and designed to bind to and protect ssDNA region at DNA replication forks during DNA replication.
  • the expression vectors for the SSB-tagged fusion proteins as described above are introduced into compatible host Cells; culture the host cells in appropriate conditions to effect the expression of the SSB-tagged fusion proteins within the host cells; lyse the cells to obtain cell lysates; contact the cell lysates with an affinity substrate immobilized with single stranded DNAs; wash off impurities; and elute the fusion proteins.
  • the term "host cell” is intended to include any cell or cell line into which a recombinant expression vector for production of a SSB-tagged fusion protein may be transfected for expression of a SSB-tagged fusion protein.
  • Suitable host cells include, but are not limited to, algal cells, bacterial cells (e.g., E. coif), yeast cells (e.g., S.cerevisiae, S. pombe), fungal cells, plant cells, invertebrate cells (e.g., insect cells such as SF9 cells, and the like), and vertebrate cells including mammalian cells.
  • the expression system includes a baculovirus vector expressed in an insect host cell.
  • An in vitro transcription and translation system with cell extracts can also be used to produce the SSB-tagged fusion proteins.
  • An expression vector encoding a SSB-tagged fusion protein can be introduced into a host cell by standard techniques such as transfection and transformation. All conventional techniques for introducing nucleic acid into host cells are included for the present invention.
  • the affinity substrate of the present invention comprises immobilized single strand DNAs that serve as the capture ligand for the SSBs in the SSB-tagged fusion proteins.
  • the capture ligand is covalently attached to or associated with a matrix material.
  • matrix materials include solids, gels, pastes, membranes, or slurries.
  • Suitable matrix materials include, but are not limited to, glass, beads, controlled pore glass, magnetic beads,' various membranes or rigid various polymeric resins such as polystyrene, polystyrene/latex, and other organic and inorganic polymers, both natural and synthetic.
  • Illustrative polymers include polyethylene, polypropylene, poly(4- methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon, nylon, polyvinyl butyrate), polyvinylidene difluoride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, and nitrocellulose.
  • Other materials that can be employed include paper, glass, minerals, ceramics, metals, metalloids, plastics, semiconductive materials, or cements.
  • substances that form gels such as proteins (e.g., gelatins), lipopolysaccharides, silicates, agarose, and polyacrylamides can be used.
  • Polymers that form several aqueous phases including, but not limited to, dextrans, polyalkylene glycols or surfactants, such as phospholipids, or long chain alkyl ammonium salts are also suitable.
  • Preferred matrix materials include resins, such as for example synthetic resins (e.g., cross-linked polystyrene, divinyl benzene, etc.), and cross-linked polysaccharides (e.g., cellulose, dextran (sephadex), agarose, sepharose), and the like.
  • the matrix material includes reactive groups capable of forming a covalent link with a SSB.
  • the matrix material includes a glyoxal activated agarose.
  • the matrix material includes a sulfhydryl reactive group.
  • the matrix material is activated with cyanogen bromide.
  • the SSBs are attached to an agarose resin by the use of a cross-linking reagent.
  • reagents are well known to those skilled in the art and include carbodiirnides, maleimides, succinimides, and reactive disulfides.
  • An affinity matrix of the present invention can take any convenient form.
  • the affinity matrix is packed into a column, a mini -column, or a capillary or microcapillary, or a capillary electrophoresis tube.
  • the affinity matrix is suspended in one phase of a multiphase solution.
  • the affinity matrix thus acts to partition the tagged molecule into that particular phase of the multiphase system.
  • Such multi-phase purification systems are well suited to large volume/high throughput applications.
  • the affinity matrix is cellulose.
  • ssDNA DNA is immobilized onto the cellulose either covalently or non-covalently; When the ssDNA is bound to the cellulose covalently, the leak or release of the bound ssDNA is minimized.
  • the ssDNA is not necessarily sequence specific.
  • double- strand DNA e.g., salmon sperm DNAs
  • dsDNA double- strand DNA
  • it is denatured first to obtain ssDNA and then immobilized onto the cellulose to produce ssDNA-cellulose affinity matrix.
  • the whole cell lysate containing the SSB-fusion proteins is applied to ssDNA-cellulose column.
  • the SSB-fusion proteins bind to immobilized ssDNA through SSB while other proteins and impurities go through the column.
  • the bound SSB-fusion protein can be simply eluted by raising salt (KC1 or NaCl) concentration.
  • salt KC1 or NaCl
  • ssDNA-cellulose affinity matrix has a capacity of at least 12mg fusion protein/ml swollen cellulose.
  • the yield of SSB-fusion protein overexpressed in E. coli is between 3 and 30 mg per liter of culture, which varies with different interest protein.
  • ssDNA-cellulose affinity matrix can be used several times for preparation of the same fusion protein or else recycled by washing with 1.5M NaCl.
  • ssDNA cellulose affinity matrix is in dry state and therefore can be kept in an indefinite time without apparent loss in both binding capacity and the efficiency of separating the SSB-fusion proteins from impurities.
  • Another aspect of the present invention provides a SSB-fusion protein comprising a SSB and an interest protein fused with the SSB either directly or indirectly.
  • the SSB-fusion protein may further comprise one or more other affinity purification tags and/or a linker for removal of the SSB partner and other affinity purification tags.
  • the SSB-fusion protein may be expressed using the expression vectors and purified using the ssDNA-irnrnobilized affinity matrix or in combination with other affinity matrix such as nickel columns as described above.
  • the fusion proteins of the present invention may be directly used in subsequent biochemical reactions since the interest proteins or polypeptides retain their antigenicity and functional activity if the SSB portion of fusion proteins does not interfere with a specific biochemical reaction.
  • the fusion protein may be cleaved to remove the SSB portion and provide just the interest proteins or polypeptides. If the production of such interest proteins or polypeptides is desired, a cleavable linker is provided in the fusion protein between the SSB and the interest proteins or polypeptides.
  • SSB bacteriophage T7
  • PCR polymerase chain reaction
  • pSSB-B 1 vector a cleavable linker (SEQ ID NO 3) recognized by the protease enterokinase was placed between the SSB and BamHl site.
  • SEQ ID NOs 4-6 the expression plasmid vectors of pSSB-B2, B3 and B4 (SEQ ID NOs 4-6) were constructed.
  • SSB bacteriophage T7
  • PCR polymerase chain reaction
  • pSSB-Yl a cleavable linker for enterokinase was placed between SSB tag and Notl site; whereas in pSSB-Y2, a cleavable linker for thrombin was placed between SSB tag and Notl site.
  • Sap 1 (SEQ ID NO 17) is an essential protein relating to DNA replication initiation process, abundant in Schizosaccharomyces pombe, and has a molecular weight of 30 kDa.
  • the DNA fragment of the sapl gene was amplified from the fission yeast genomic DNA by PCR reaction and inserted into pSSB-B4 vector at BamHI and Hindlll sites.
  • the recombinant plasmid encoding for the fusion protein 6His-SSB-Sapl was transformed into E. coli BL21(DE3)pLys cells to obtain individual colonies.
  • a single colony was introduced into a 50 ml of liquid LB medium containing 20 ⁇ 3 ⁇ 4/ ⁇ 1 of Kanamycin and incubated for overnight.
  • IPTG was added to 0.1 mM and the culture was incubated for additional 3.5 hrs.
  • the cells were then recovered by cehtrifugation, resuspended in the buffer A (50 mM Tris-HCl, pH 7.4, 5 mM Magnesium Acetate, 5 mM DTT, 1 mM EDTA, 1 mM EGTA, 0.04% NP-40, 10% glycerol) containing 0.4M KC1, broken by sonication, and spun at 37,000g for 30 min.
  • the whole cell extract and subsequent pellet and supernatant of the cell lysate were examined by SDS-PAGE electrophoresis.
  • the supernatant portion containing 6His-SSB-Sapl was adjusted to salt concentration of 0.2 M with buffer A and then applied to ssDNA-cellulose column. The column was washed with buffer A containing 0.2M KC1 to remove unbound proteins and other impurities. Then, the bound fusion protein 6His-SSB-Sapl was eluted at -0.4 M KG. As shown in FIG 3 A, after one step of ssDNA-cellulose chromatography the purity of 6His-SSB-Sapl reached to -96% as measured by densitometry.
  • 6His-SSB-Sapl purified by ssDNA-cellulose affinity chromatography was applied to Ni 2+ -NTA agarose column. As indicated in FIG 3B, 6His-SSB-Sapl bound to Ni 2 t column well and was eluted at 250 mM imidazole. This indicates that 6His-SSB-tagged fusion proteins may be purified with two chromatographic columns of different characteristics. One is ssDNA-cellulose column and the other is Ni 2+ -NTA agarose column. By the combination of ssDNA-cellulose and Ni 2+ -agarose chromatographic columns, 6His-SSB-tagged fusion proteins that are expressed even at low level can be generally purified to high homogeneity for subsequent biochemical characterization.
  • Fenl gene (SEQ ID . NO 18) was inserted into pSSB-Bl vector at Sad and Hindlll sites.
  • the fusion protein SSB-Fenl was overexpressed in E, coli BL21(DE3)pLys cells.
  • SSB-Fenl was soluble in buffer A containing 0.1 M KCl.
  • SSB-Fenl bound to ssDNA-cellulose well and was eluted at -0.4 M KCl.
  • the purity of SSB-Fenl reached to -96% as measured by densitometry.
  • Both of 6His-GST-Sapl and 6His-Sapl are soluble in cell extracts containing 0.4M C1.
  • the cell extract containing 6His-GST-Sapl or 6His-Sapl was adjusted to salt concentration of 0.2 M with buffer A and then applied to Glutathione Sepharose 4B or Ni 2+ -NTA agarose column, respectively.
  • the columns were washed with buffer A containing 0.2M KC1 to remove unbound proteins and other impurities.
  • the column was further washed with buffer A containing 0.2M KC1 and 20 mM imidazole.
  • the bound 6His-GST-Sapl was first eluted with 10 mM glutathione and the remaining 6His-GST- Sapl was eluted with 0.5% SDS.
  • the purity of 6His-GST-Sapl reached to -20% and -70% when it was eluted by 10 mM glutathione and 0.5% SDS, respectively.
  • the result shown in FIG 6 indicated that the 6His-Sapl was eluted at 250 mM imidazole and its purity reached to -70%.
  • the purity of the fusion protein 6His-SSB-Sapl reached to -96% after ssDNA-cellulose affinity chromatography.
  • SSB-fusion proteins have been overproduced in E. coli, yeast, insect, or human cells. No matter whether the interest proteins or polypeptides are from prokaryotic or eukaryotic cells, all of these fusion proteins achieved good solubility.
  • the overproduced SSB-fusion proteins are applied to ssDNA cellulose and all of them retain in the ssDNA-cellulose column well, indicating that the SSB fused to interest proteins or polypeptides maintains its capability of binding to ssDNA and the interest proteins or polypeptides do not spatially hinder the binding of SSB to ssDNA, which guarantees the good separation of SSB-fusion proteins from impurities through ssDNA-cellulose affinity chromatography.
  • All SSB-fusion proteins were designed to contain a cleavage site at the junction of SSB and interest proteins.
  • the cleavage site permits the generation of interest proteins or polypeptides without the fused SSB.
  • All purified SSB-fusion proteins are good substrates for cleavage by site-specific proteases such as thrombin or enterokinase.
  • the protease used is one that does not cleave the SSB and the interest protein.
  • the SSB can be removed by absorption to ssDNA-cellulose or Ni 2+ -agarose column if six histidines are fused to the N-terminus or C -terminus of the SSB.

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Abstract

La présente invention concerne un vecteur d'expression comprenant un promoteur et une séquence polynucléotidique codant pour une protéine de fusion. La présente invention concerne en outre un procédé de purification d'une protéine d'intérêt. La présente invention concerne également une protéine de fusion comprenant une protéine de liaison à l'ADN simple brin et une protéine d'intérêt ou un polypeptide fusionné directement ou indirectement à l'extrémité COOH-terminale ou à l'extrémité NH2-terminale de la protéine de liaison à l'ADN simple brin, ladite protéine de fusion pouvant se lier à de l'ADN simple brin.
PCT/CN2010/002000 2009-12-16 2010-12-09 Protéine de fusion contenant une protéine de liaison à l'adn simple brin et procédés d'expression et de purification associés WO2011072479A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023072145A1 (fr) * 2021-10-26 2023-05-04 南京金斯瑞生物科技有限公司 Procédé de purification d'adn simple brin

Families Citing this family (3)

* Cited by examiner, † Cited by third party
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CN101709306B (zh) * 2009-12-16 2014-07-09 孔道春 含单链dna结合蛋白的融合蛋白及其表达和纯化的方法
CN112812192B (zh) * 2021-01-22 2022-05-20 湖南大学 一种作为核酸-抗体共缀物通用载体的ProA/G-dRep融合蛋白及其应用
CN113278642A (zh) * 2021-04-30 2021-08-20 上海交通大学 一种深海古菌单链dna结合蛋白ssb及其制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070059713A1 (en) * 2005-09-09 2007-03-15 Lee Jun E SSB-DNA polymerase fusion proteins
CN101709306A (zh) * 2009-12-16 2010-05-19 孔道春 含单链dna结合蛋白的融合蛋白及其表达和纯化的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100457911C (zh) * 2006-01-24 2009-02-04 中国人民解放军第二军医大学 含有效b细胞活化因子基因启动子的重组质粒及其制备方法和用途
CN101161287A (zh) * 2006-10-12 2008-04-16 复旦大学附属妇产科医院 一种携人分子佐剂的融合蛋白避孕疫苗及其制备方法
CN101067138A (zh) * 2007-03-30 2007-11-07 中国人民解放军第三军医大学第一附属医院 基因工程制备人毛乳头细胞hspc016蛋白质的方法、表达载体和工程菌

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070059713A1 (en) * 2005-09-09 2007-03-15 Lee Jun E SSB-DNA polymerase fusion proteins
CN101709306A (zh) * 2009-12-16 2010-05-19 孔道春 含单链dna结合蛋白的融合蛋白及其表达和纯化的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 14 July 2008 (2008-07-14), Database accession no. AZ32836 *
HANSJORG LEHNHERR ET AL.: "Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1", JOURNAL OF BACTERIOLOGY, 31 October 1999 (1999-10-31), pages 6463 - 6468 *
QIAO HUI-LI ET AL.: "Clon ing, High Expression of Single-Stranded DNA-Binding Protein and Its Interaction with ssDNA", CHINA BIOTECHNOLOGY, vol. 27, no. 4, 31 December 2007 (2007-12-31), pages 12 - 17 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023072145A1 (fr) * 2021-10-26 2023-05-04 南京金斯瑞生物科技有限公司 Procédé de purification d'adn simple brin

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