WO2006078273A2 - Procedes et compositions permettant de produire des proteines recombinees - Google Patents

Procedes et compositions permettant de produire des proteines recombinees Download PDF

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WO2006078273A2
WO2006078273A2 PCT/US2005/013553 US2005013553W WO2006078273A2 WO 2006078273 A2 WO2006078273 A2 WO 2006078273A2 US 2005013553 W US2005013553 W US 2005013553W WO 2006078273 A2 WO2006078273 A2 WO 2006078273A2
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protein
fusion
fragment
proteins
host cell
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PCT/US2005/013553
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WO2006078273A3 (fr
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Deb K. Chattergee
Dominic Esposito
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The United States Of America As Represented By Teh Secretary Of Health And Human Services, Nih
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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
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin

Definitions

  • This invention relates to methods and compositions for producing recombinant proteins, especially in high yield.
  • the invention is particularly amenable to producing proteins that are insoluble or minimally soluble in aqueous buffers.
  • the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a solubility facilitating protein.
  • E. coli expression system offers many advantages. It is easy to handle, cost-effective, and can produce proteins in high yield (Schmidt, M. et al. (2002) “EXPRESSION SYSTEMS FOR PRODUCTION OF RECOMBINANT ALLERGENS,” Int Arch Allergy Immunol. 128(4):264-270). Unfortunately, however, such enhanced production is frequently accompanied by problems of protein insolubility, production host non-viability, and aberrant protein folding (Zhang, Z. et al.
  • additional "affinity tag" peptide sequences such as HiS 6 , thioredoxin, nusA, glutathione S-transferase (GST),
  • Strep-tag Strep-tag, FLAG ® (Sigma Aldrich), AviTag, calmodulin-binding peptide (CBP), etc.
  • CBP calmodulin-binding peptide
  • Such sequences include affinity "tags” such as FLAG, c-myc, S-tag, AviTag, and His 6 (Ashraf, S. S. et al. (2004) “A NOVEL MULTI-AFFINITY TAG SYSTEM TO PRODUCE HIGH LEVELS OF SOLUBLE AND BIOTINYLATED PROTEINS IN ESCHERICHIA COLI,” Protein Expr Purif. 33(2):238-245; Terpe, K. (2003) “OVERVIEW OF TAG PROTEIN FUSIONS: FROM MOLECULAR AND BIOCHEMICAL FUNDAMENTALS TO COMMERCIAL
  • PROTEINS STRUCTURES, FUNCTIONS AND THEIR APPLICATION IN GENE ENGINEERING FOR EXPRESSING HETEROLOGOUS PROTEINS IN ESCHERICHIA COLI] (ENGLISH TRANSLATION)" Sheng Wu Gong Cheng Xue Bao. 18(3):261-266).
  • the chaperone fusions used by these researchers contained a signal sequence that directs the fusion protein to the periplasmic space. Such expression has been found to be problematic. Some proteins have difficulty traversing the cytoplasmic membrane, can aggregate in the periplasm, and can contribute to cell toxicity issues. To address these problems, Levy, R. el al.
  • coli cytoplasm is normally maintained at a low redox potential, and as a result, oxidative protein folding (such as that involved in disulfide bond formation) does not normally occur there.
  • oxidative protein folding such as that involved in disulfide bond formation
  • the use of specialized (trxB ⁇ gor ' ) host cells is taught (Levy, R. et al. (2001) "PRODUCTION OF CORRECTLY FOLDED FAB ANTIBODY FRAGMENT IN THE CYTOPLASM OF ESCHERICHIA COLI TRXB GOR MUTANTS VIA THE COEXPRESSION OF MOLECULAR CHAPERONES," Protein Expr Purif. 23(2):338- 347).
  • FIG. 1 shows the expression of total (T) and soluble (S) proteins Wnt5A, Folliculin, and IFN-Hyb3 using a Skp fusion expression system.
  • FIG. 1 shows the expression of total (T) and soluble (S) proteins YopD and Endostatin using a Skp fusion expression system.
  • Figure 3 shows the expression of total (T) and soluble (S) proteins Hifl A, IL-13, a domain of Folliculin (FD) and full-length Folliculin (F) using a Skp fusion expression system.
  • FIG. 4 shows the expression of total (T) and soluble (S) proteins Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin using a MBP fusion expression system.
  • Figure 5 shows the expression of total (T) and soluble (S) proteins Folliculin, IFN-Hyb3, and Wnt5a using a DsbC fusion expression system.
  • Figure 6 illustrates the positions of the genetic determinants of Skp fusion plasmid pDest 579, and the DsbC fusion plasmid pDest-568.
  • FIG. 7 demonstrates that Folliculin is highly insoluble when expressed in E. coli even in the presence of various chaperones, GroEL- ES/Trigger factor (TF) or Skp/DsbC. Arrows indicate the position of the chaperones or the fusion proteins.
  • Figure 8 illustrates the configuration of preferred fusion proteins of the Protein Kinase Protein (PK) and a Target Protein.
  • PK Protein Kinase Protein
  • AF Affinity Tag.
  • Figure 9 illustrates the ability of the PK fusion protein to facilitate the solubility of Hifl a, Folliculin, a Folliculin domain (FD) and ILl 3.
  • An amino terminal PK fusion is used. Arrows indicate the position of fusion proteins.
  • Figure 10 shows the purification of Strep Il-PK-Folliculin using Strep- tactin.
  • FIG. 11 shows the purification of PK-DEFG Folliculin using Strep- tactin.
  • Figure 12 shows TEV protease cleavage of Folliculin and Folliculin domain fusion.
  • Figure 13 shows a comparison of the level of expression and solubility of Folliculin obtained with StrepII-Skp or Strepll-PK fusion partners with Tev5 and TevlO Fusions.
  • Figure 14 shows the purification of Strep-tag II-PK-Folliculin TevlO fusion protein.
  • Figure 15 shows TEV protease digestion of a Strepll-PK-TEV 10- Folliculin fusion molecule.
  • the present invention thus relates to recombinant methods and compositions for producing recombinant proteins, especially in high yield.
  • the invention is particularly amenable to producing proteins that are typically insoluble or minimally soluble in aqueous buffers when produced by other recombinant methods.
  • the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a chaperone protein, to a phage T7 protein kinase, or to a fragment thereof.
  • Recombinant proteins and their fragments are produced in E. coli or other hosts for a variety of reasons such as basic scientific research, structure determinations, clinical or therapeutic applications, etc.
  • solubility facilitating proteins are fused to a desired target protein (to form an "expression tag") and employed to facilitate the expression of the target protein.
  • a desired target protein to form an "expression tag”
  • solubility facilitating proteins may comprise a chaperone protein. Chaperone proteins are expressible in high yield, and help to make proteins fold correctly.
  • the solubility facilitating proteins of the present invention may comprise the phage T7 protein kinase, or a fragment thereof.
  • polynucleotides encoding such solubility facilitating proteins are fused to the N- or C-terminus of a polynucleotide encoding a desired target protein (either directly, or through a linking sequence).
  • a chaperone protein is employed as the solubility facilitating protein
  • such polynucleotides will not encode the chaperone protein's signal sequence.
  • the expressed proteins are retained in the cytoplasm rather than the periplasm. Such cytoplasmic retention prevents possible interference with cell viability that might occur if the expressed proteins were secreted into the periplasm.
  • any of a variety of suitable chaperone proteins may be employed in concert with the present invention, the use of the signal sequence-less chaperone proteins: Skp, DsbC in such "Expression Tags" is particularly preferred.
  • additional sequences e.g., poly-His, Strep-tag or Maltose Binding Protein (MBP) are preferred.
  • the invention provides a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein, (especially a chaperone protein, or a phage T7 protein kinase, or a fragment thereof), linked to a desired target protein.
  • a solubility facilitating protein especially a chaperone protein, or a phage T7 protein kinase, or a fragment thereof
  • the invention particularly concerns the embodiment of such a nucleic acid molecule wherein the solubility facilitating protein comprises a fragment of the chaperone protein or the phage T7 protein kinase.
  • the invention further concerns the embodiments of such nucleic acid molecules wherein the encoded fragment of the chaperone protein lacks a signal sequence.
  • the invention concerns the embodiments of such nucleic acid molecules wherein the chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein.
  • the invention concerns the embodiments of such nucleic acid molecules wherein the solubility facilitating protein is the phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the solubility facilitating protein is the phage T7 protein kinase, or a fragment thereof
  • the fusion protein additionally comprises an affinity tag
  • the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the invention concerns the embodiments of such nucleic acid molecules wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the invention further concerns a method of producing a desired target protein, wherein the method comprises the steps:
  • a plasmid vector capable of being expressed in a host cell comprising a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein (especially a chaperone protein, a phage T7 protein kinase, or a fragment of either), linked to the desired target protein;
  • a solubility facilitating protein especially a chaperone protein, a phage T7 protein kinase, or a fragment of either
  • the solubility facilitating protein comprises a fragment of a chaperone protein, and/or wherein such fragment lacks a signal sequence
  • the chaperone protein is selected from the group consisting of E. coli DsbC protein and E coli Skp protein
  • the fusion protein additionally comprises an affinity tag
  • the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the invention further concerns the embodiments of such method wherein the solubility facilitating protein comprises a fragment of the phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the solubility facilitating protein comprises a fragment of the phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the invention further concerns a desired target protein produced by the process of:
  • a plasmid vector capable of being expressed in a host cell comprising a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises a solubility facilitating protein (especially, a chaperone protein, a phage T7 protein kinase, or a fragment of either), linked to the desired target protein;
  • a solubility facilitating protein especially, a chaperone protein, a phage T7 protein kinase, or a fragment of either
  • the invention further concerns the embodiments of such method wherein the solubility facilitating protein comprises a fragment of a chaperone protein, and/or wherein such fragment lacks a protein signal sequence, and/or wherein the chaperone protein is selected from the group consisting of E. coli DsbC protein and E. coli Skp protein, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the invention further concerns the embodiments of such method wherein the fusion protein encodes a phage T7 protein kinase, or a fragment thereof, and/or wherein the fusion protein additionally comprises an affinity tag, and/or wherein the nucleic acid molecule is a plasmid vector capable of replicating in a host cell (especially wherein the host cell is an E. coli host cell).
  • the present invention relates to recombinant methods and compositions for producing recombinant proteins, especially in high yield.
  • the invention is particularly amenable to producing proteins that are typically insoluble or minimally soluble (i.e., where less than 50% and more preferably if less than 30%, still more preferably less than 20%, and most preferably less than 10% of total protein produced is insoluble) in aqueous buffers when produced using other recombinant methods.
  • the invention concerns the use of fusion molecules in which a desired protein is produced as a fusion molecule linked to a solubility facilitating protein, especially a chaperone protein or the phage T7 protein kinase.
  • the invention particularly pertains to the use of the E.
  • coli DsbC protein the E. coli Skp protein, or a protein derived from such proteins (e.g., a truncated form of such proteins, etc.) as chaperone proteins (e.g., fusion partners) to mediate the enhanced expression of the target proteins, and to the use of the phage T7 protein kinase, or a protein derived therefrom (e.g., a truncated form of the phage T7 protein kinase, etc.) as a fusion partner to mediate the enhanced expression of the target proteins.
  • fusion molecules of such proteins are used in conjunction with affinity tags (e.g., proteins or polypeptides that can serve to facilitate the recovery or purification of the expressed fusion protein).
  • the present invention relates to the use of gene fusions to improve the yield and to address the insolubility of desired target proteins produced through the use of recombinant DNA techniques.
  • a "target" protein is any recombinant protein whose production and/or recovery is desired.
  • Target proteins may comprise any protein including enzymes, hormones, cytokines, growth factors, etc.
  • Such proteins may be of prokaryotic origin (e.g., bacterial, viral, etc.) or eukaryotic (e.g., fungal, mammalian (especially human), etc.).
  • the present invention particularly pertains to the use of Escherichia coli as a host cell for protein production. As will be appreciated, however, the principles of the present invention may be readily adapted to permit the use of the present invention in other host cells.
  • fusion polynucleotides encoding the target protein are operably linked to polynucleotides encoding one or more solubility facilitating proteins, or one or more functional domains thereof, are employed.
  • Such fusion polynucleotides possess codons encoding the solubility facilitating protein (or domain(s) thereof) in frame with those encoding the target protein, so that, upon expression in a host cell, the fusion polynucleotide will mediate the production of a fusion protein comprising the solubility facilitating protein (or domain(s) thereof) and the target protein.
  • solubility facilitating protein is intended to refer to a protein that enhances the solubility of a desired target protein to which it is fused.
  • Preferred solubility facilitating proteins include the phage T7 protein kinase and chaperone proteins. Suitable chaperone proteins are disclosed by Lund, P.A. (2001 ) "MICROBIAL MOLECULAR CHAPERONES,” Adv Microb Physiol. 44:93-140; Bardwell, J.C. et al. (1993) "THE BONDS THAT TIE: CATALYZED DISULFIDE BOND FORMATION," Cell 74(5):769-771 ; Ellis, R.J.
  • the invention particularly concerns the embodiments in which the solubility facilitating protein domain(s) are selected from the group consisting of heat shock proteins (HsP), DsbC, Skp or MBP chaperone proteins, or the phage T7 protein kinase, or proteins, or domains thereof, derived therefrom.
  • HsP heat shock proteins
  • DsbC DsbC
  • Skp or MBP chaperone proteins or the phage T7 protein kinase, or proteins, or domains thereof, derived therefrom.
  • protein kinase a fusion partner derived from a phage T7 protein
  • This protein has been shown to stimulate its own expression, and the stimulation occurs at both translational as well post-transcriptional levels.
  • the level of expression of this protein itself is extremely high as it synthesizes up to 40% of the total E .coli protein even from a single copy gene. It is also an extremely soluble protein.
  • the polynucleotide encoding the solubility facilitating protein domain(s) may be fused to the polynucleotide encoding the target protein in any of several orientations.
  • the polynucleotide encoding a chaperone protein or the T7 protein kinase, or a domain thereof may be linked to that which encodes either the amino or carboxyl terminus of the target protein.
  • T7 protein kinases or domains thereof (which may be the same or different from one another, or combinations thereof) may be used in a single fusion molecule; likewise, multiple target molecules (which may comprise multiple copies of the same target molecule, or multiple different target molecules, or combinations thereof) may be used in a single fusion molecule.
  • Certain chaperone proteins possess signal sequences that serve to direct the expressed protein out of the cytoplasm and into the periplasm of a host cell.
  • the fusion polynucleotide employed will contain the signal sequence(s) that are sufficient to mediate such transport.
  • the fusion polynucleotide will encode only a fragment of the chaperone protein, and will not contain the chaperone protein's signal sequence.
  • the expressed protein will accumulate in the cytoplasm. The use of this embodiment is preferred for enhancing the production and/or recovery of proteins whose accumulation in the periplasm might affect cellular viability.
  • the fusion polynucleotides of the invention may contain one or more additional polynucleotides in addition to those encoding the solubility facilitating protein or protein domain(s) and the target protein.
  • the present invention contemplates that such fusion polynucleotides may contain polynucleotides that, upon expression, encode proteins or polypeptides that can serve to facilitate the recovery or purification of the expressed fusion protein.
  • such fusion polynucleotides may encode a poly-His peptide (e.g., HiS 6 ), poly-Arg peptide (e.g., Arg 6 ), streptavidin binding protein (SBP), nusA, TrxA, DsbA, calmodulin-binding peptide (CBP), calmodulin-binding domain (CBD), glutathione S-transferase (GST), FLAG ® (Sigma Aldrich), AviTag, chitin binding domain, etc. (see, Terpe, K.
  • poly-His peptide e.g., HiS 6
  • poly-Arg peptide e.g., Arg 6
  • streptavidin binding protein SBP
  • nusA nusA
  • TrxA calmodulin-binding peptide
  • DsbA calmodulin-binding peptide
  • CBD calmodulin-binding domain
  • GST glutathione S-transferase
  • Such additional polynucleotide(s) may be placed before or after the solubility facilitating protein-encoding sequences, and before or after the polynucleotide sequences that encode the desired target protein.
  • the methods and compositions of the present invention are useful for producing catalytic proteins, such as enzymes, co-factors, etc. that may be used to catalyze chemical or biochemical reactions.
  • Such proteins include restriction endonucleases, polymerases, exonucleases, proteases, peptidases, amylases, xylanases, cellulases, chitinases, lipases, hydrolases, hydrogenases, dehydrogenases, etc. (see, Haki, G.D. et al. (2003) "DEVELOPMENTS IN INDUSTRIALLY IMPORTANT THERMOSTABLE ENZYMES: A REVIEW,” Bioresour Technol. 89(1): 17-34; Panke, S.
  • the methods and compositions of the present invention are further useful for producing diagnostic proteins, such as antigens, haptens, single chain antibodies, etc. (see, Blazek, D. et al. (2003) “THE PRODUCTION AND APPLICATION OF SINGLE-CHAIN ANTIBODY FRAGMENTS," Folia Microbiol (Praha).48(5):687- 698; Bilbao, G. et al.
  • the methods and compositions of the present invention may be used to produce diagnostic compositions useful in the diagnosis of diseases such as cancer, Alzheimer's disease, Parkinson disease, diabetes, inflammatory and autoimmune diseases, anemia, AIDS, SARS, influenza, etc.
  • the methods and compositions of the present invention are additionally useful for producing therapeutic proteins, such as humanized antibodies, albumins, hormones (e.g., insulin, growth hormone, etc.), receptors (e.g., adrenocorticotropic hormone receptor and its bioactive fragments, angiotensin receptor, atrial natriuretic receptor, bradykininin receptor, growth hormone receptor, chemotatic receptor, dynorphin receptor, endorphin receptor, the receptor for ⁇ -lipotropin and its bioactive fragments, enkephalin receptor, enzyme inhibitor receptors, the receptor for fibronectin and its bioactive fragments, gastrointestinal- and growth hormone-releasing peptide receptors, the receptor for luteinizing hormone releasing peptide, the receptor for melanocyte stimulating hormone, neurotensin receptor, opioid receptor, oxytocin receptor, vasopressin receptor, vasotocin receptor, the receptor for parathyroid hormone and fragments, protein kinase receptor, somatostatin
  • compositions of the present invention may be used to produce pharmaceutical compositions useful in therapies for diseases such as cancer, Alzheimer's disease, Parkinson disease, diabetes, inflammatory and autoimmune diseases, anemia, AIDS, SARS, influenza, etc.
  • E. coli strains BL21 (DE3), E. coli BL21 (Rosetta), DB3.1 and DH5 ⁇ are all commercially available from Novagen and Invitrogen, respectively.
  • BL21 has the genotype: F " ompT [lo ⁇ hsdS ⁇ ( ⁇ B ITI B ; an E. coli B strain) with DE3, a ⁇ prophage carrying the T7 RNA polymerase gene.
  • DH5 ⁇ has the genotype: F “ , ⁇ S0dlacZM15, endAl, recAl, hsdRl 7 (rk ⁇ , mk + ), supE44, thi- ⁇ , gyrA96, relAl, A ⁇ lacZYA-argF)UJ69, ⁇ " .
  • Plasmid pET43-DVbase is constructed by digesting pET43a (Novagen) with Ndel and Hindlll, and then inserting (via ligation) a DNA fragment created by annealing the two oligonucleotide primers SEQ ID NO:1 and SEQ ID NO:2.
  • pDest-590 pET43-DVbase is then digested with EcoRV, and the Gateway rfa cassette (Invitrogen) was ligated into the vector. The ligation mixture is then transformed into E. coli DB3.1 (Invitrogen) and selected on 100 ⁇ g/ml ampicillin and 15 ⁇ g/ml chloramphenicol.
  • Additional amino-terminal fusion destination vectors are constructed by digestion of pDest-590 with BgIIl, and ligation of BamHl digests of various PCR fragments for the different fusion proteins.
  • the primer sequences used to amplify the fusion proteins for these vectors were: pDest-566 (His ⁇ -MBP)
  • This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the maltose binding protein of E. coli (Genbank Accession No. JOl 648; Roa, M. et al. (1980) "LOCATION OF A PHAGE BINDING REGION ON AN OUTER MEMBRANE PROTEIN," FEBS Lett. 121 (1): 127- 129; Bedouelle, H. et al ( 198O) 11 MUTATIONS WHICH ALTER THE FUNCTION OF THE SIGNAL SEQUENCE OF THE MALTOSE BINDING PROTEIN OF ESCHERICHIA COLL” Nature 285 (5760):78-81 ; Emr, S.D.
  • pDest-566 (His6-MBP) is produced from pDest590 by ligating a BamHI digested PCR fragment containing the E. coli MBP gene.
  • the MBP gene fragment was obtained from E. coli DNA via PCR using the primers:
  • Reverse primer (SEQ ID NO:4) ccacccaccg gatcccgaat tagtctgcgc gtctttcagg gcttc
  • This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the E. coli DsbC gene (Genbank Accession No. U28375).
  • the amino acid sequence of the DsbC protein is (SEQ ID NO:5): MKKGFMLFTL LA ⁇ FSGFAQA DDAAIQQTLA KMGIKSSDIQ PAPVAGMKTV
  • SEQ ID NO:5 contains the mature DsbC protein as well as the signal sequence responsible for mediating the transfer of the protein to the periplasm.
  • pDest-568 is produced from pDest590 by ligating a BamHI digested PCR fragment containing the DsbC gene (including its signal sequence).
  • the E. coli DsbC fragment was obtained from E. coli DNA via PCR using the primers:
  • Reverse primer (SEQ ID NO:7) cgagttagag gatcctttac cgctggtcat tttttggtgt teg [0063
  • This vector is capable of forming a fusion protein between a desired target gene, and gene sequences that encode the E. coli skp gene (Genbank Accession No. M21 1 18; Hoick, A. et al. (1988) "CLONING AND SEQUENCING OF THE GENE FOR THE DNA-BiNDiNG 17K PROTEIN OF ESCHERICHIA COLI," Gene 67 (1 ): 1 17- 124).
  • the amino acid sequence of the Skp protein is (SEQ ID NO:9):
  • pDest-579 (signal sequence-less Skp) is produced from pDest590 by ligating a BamHl digested PCR fragment containing the E. coli Skp gene (minus its signal sequence).
  • the Skp fragment was obtained from E. coli DNA via PCR using the primers: Forward primer (SEQ ID NO:10) gcgagcgagg atccgctgac aaaattgcaa tcgtcaacat ggg
  • Reverse primer (SEQ ID NO: 11) aggctagcgg atcctttaac ctgtttcagt acgtcggcag
  • DNA is transformed into E. coli DB3.1 , selected on ampicillin and chloramphenicol, and plasmids are isolated from individual clones. These plasmids are then sequenced completely throughout the fusion protein to verify that the correct clone is generated in the correct orientation.
  • Expression vectors with Skp, DsbC and MBP (maltose binding protein) proteins are made by introducing gene sequences encoding desired target proteins into the above-described destination vectors using standard recombination mediated cloning procedures (Invitrogen). For Skp, signal sequence is eliminated so that expression fusion proteins will be retained in the cytoplasm.
  • H. sapiens B ⁇ D (Folliculin): AF517523 Forward primer (SEQ ID NO:13): ggggacaact ttgtacaaaa aagttggcac catgaatgcc atcgtggctc tctgccac
  • Reverse primer (SEQ ID NO:14): ggggacaact ttgtacaaga aagttggcta gttccgagac tccgaggctg tggggc
  • H. sapiens Wnt5a NM_003392 Forward primer (SEQ ID NO: 15): aggtggctcg ggtgctggcc aggttgttat agaagctaat tc
  • Reverse primer (SEQ ID NO:16): ggggacaact ttgtacaaga aagttggcta tttgcacacg aactgatcca caatc
  • H. sapiens Endostatin AFl 84060 Forward primer (SEQ ID NO: 18): ggcgaaaacc tgtacttcca aggccacagc caccgcgact tccagccggt gc
  • Reverse primer (SEQ ID NO: 19): ggggacaact ttgtacaaga aagttggcta cttggaggca gtcatgaagc tg
  • Y. pestis YopD CAB54905
  • Reverse primer (SEQ ID NO:22): ggggacaact ttgtacaaga aagttggcta gacaacacca aaagcggctt tcatgg
  • Reverse primer (SEQ ID NO:25): ggggacaact ttgtacaaga aagttggcta gttaacttga tccaaagctc tgag
  • Reverse primer (SEQ ID NO:28): ggggacaact ttgtacaaga aagttggcta cgcgttgaaa cgaccttcac gg
  • Adapter Primer (SEQ ID NO:29): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc
  • Reverse primer (SEQ ID NO:31): ggggacaact ttgtacaaga aagttggtta ttccttcctc cttaatcttt cttg
  • Adapter primer (SEQ ID NO:32): ggggacaact ttgtacaaaa aagttggcga aaacctgtac ttccaaggc
  • pDest 566 maltose binding protein-fusion vector
  • pDest 579 Skp-Fusion vector
  • pDest 568 DsbC fusion vector
  • genes are amplified from cDNA or genomic DNA using primers containing Gateway recombination signal sequences.
  • a TEV protease cleavage site is introduced in front of the gene of interest to allow cleavage of the fusion protein after expression.
  • a second "adapter" primer is employed during amplification.
  • the start and stop primers are added in the initial PCR reaction (200 nM each in a 50 ⁇ l reaction), along with 100-200 ng of template DNA. After 5 cycles of amplification, 200 nM adapter primers are added to the reactions, and amplification is continued for an additional 15 cycles. In cases where no adapter primer is needed, PCR is carried out for 15 cycles total. PCR cycling conditions are: 95°C, 30 sec; 55°C, 30 sec, 72 0 C, 1 min per Kb product. After amplification, the PCR products are cleaned using Qiagen's PCR Purification columns, and DNA is introduced into a Gateway BP reaction.
  • 1 ⁇ l of PCR product is mixed with 150 ng of pDonr223 vector (Invitrogen) in a 20 ⁇ l reaction, and reacted with 4 ⁇ l BP Clonase for 1 hour at 30 °C. After stopping the reaction with Proteinase K, 1 ⁇ l is transformed into E. coli DH5a and samples were plated on LB with 50 ⁇ g/ml spectinomycin to select for correct clones. Several clones are grown, and plasmid DNA is prepared by alkaline lysis or FastPlasmid (Brinkmann). Clones are then sequence verified to ensure that no mutations had been introduced.
  • genes encoding Folliculin, Wnt5a, IFN- Hyb3, Endostatin, YopD, Hifl ⁇ , IL- 13 and a Folliculin domain are cloned into the Skp fusion destination vector
  • genes encoding Folliculin, Wnt5a, IFN-Hyb3, Endostatin, YopD and a Folliculin domain are cloned into the MBP fusion destination vector
  • genes encoding Folliculin, Wnt5a and IFN-Hyb3 are cloned into the DsbC fusion destination vector.
  • Cloning is accomplished according to the protocol of Gateway LR reaction (Invitrogen). A portion of the reaction (1 ⁇ l) is used to transform E. coli DH5 ⁇ and plated onto ampicillin containing plates. Individual clones are digested with BsrGI to check for correct insert size. Correct clones were saved.
  • a single colony of each clone is grown in Circle Grow growth medium (Q- Biogene, California)) with amp (100 ug/ml) and chloramphenicol (15 ⁇ g/ml) at 30 0 C for 15 hrs.
  • 450 ⁇ l of an overnight grown culture is inoculated into 15 ml Circle Grow (Q-Biogene) media containing ampicillin (100 ⁇ g/ml) and chloramphenicol (15 ⁇ g/ml) and grown at 30°C for approximately 4 hrs to an As 60 of 0.7-0.8.
  • the culture is induced with 1 mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG) for 3 hr. at 3O 0 C and then harvested by centrifugation.
  • IPTG isopropyl- ⁇ -D- thiogalactopyranoside
  • Cells from the centrifuged 15 ml culture are suspended in 1 ml of Buffer (50 mM Tris, pH8, 50 mM NaCl). 100 ⁇ l of protease inhibitor (Roche, 1 tablet dissolved in 600 ⁇ l of the Buffer) is added to the cell suspension before sonication with a micro-tip (3 times for 10 sec each at 65% efficiency using a micro-tip probe). A portion (50-100 ⁇ l) is saved to permit a determination of total (T) protein expression. The remaining material is centrifuged for 5 min at maximum speed (14,000-15,000 rpm) in a cold microcentrifuge (Eppendorf). The supernatant is saved and analyzed for soluble (S) expression of the test protein. Usually, 1-1.5 ⁇ l of the sample is used for polyacrylamide gel analysis (4-20%, Invitrogen). The gel is stained with Coomassie blue.
  • Skp fusion vectors are prepared and used to express Wnt5a, Folliculin, IFN-Hyb3, Endostatin and YopD as Skp fusions.
  • BL21 DE3 (Rosetta) containing the plasmid is grown as described in the Example 1.
  • Figure 1 shows the results of efforts to express Wnt5a, Folliculin and IFN-Hyb3 as Skp fusions.
  • Wnt5a with Skp fusion produces almost 40% of the total protein of which approximately 15% is soluble.
  • Folliculin is expressed extremely well as almost 30% of the total protein.
  • the soluble fraction of Skp-folliculin is estimated to be about 40% of total Folliculin protein.
  • total amount of expressed protein was about 25% of which about 25% was soluble.
  • FIG. 2 shows the expression of YopD and Endostatin as Skp fusion.
  • YopD is expressed as 5-10% of total protein of which about 30-40% is soluble.
  • Skp-Endostatin fusion produces almost 50% of total protein. However, about 15% is soluble.
  • Figure 3 shows the expression of total (T) and soluble (S) proteins Hifl A, IL-13, a domain of Folliculin (FD) and full-length Folliculin (F) using a Skp fusion expression system.
  • signal sequence-less Skp fusion of IL- 13 and a domain of Folliculin produces 40 and 50% of total protein, respectively.
  • about 30% of ILl 3 and 25% of Folliculin domains are soluble.
  • a repeat experiment with Folliculin produces the same result as shown in Figure 1. The only protein that is produced in almost 100% insoluble form was Hifl ⁇ , although the level of expression is quite good.
  • MBP fusion vectors are prepared and used to express, Folliculin, IFN-Hyb3, Wnt5a YopD and Endostatin as MBP fusions.
  • Figure 4 shows the results of efforts to express Folliculin, IFN- Hyb3, Wnt5a, YopD and Endostatin as MBP-fusion.
  • the level of expression of Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin are 15%, 20%, 20%, 20% and 40%, respectively.
  • the amount of soluble fusion protein for Folliculin, IFN-Hyb3, Wnt5a, YopD and Endostatin is estimated to be about 40%, 25%, 5%, 50% and 30%, respectively.
  • DsbC fusion vectors are prepared and used to express, Folliculin, 1FN-Hyb3, and Wnt5a as DsbC fusions.
  • the fusion protein will be accumulated in the periplasmic space.
  • Figure 5 shows the results of efforts to express Folliculin, IFN-Hyb3 and Wnt5a as DsbC fusions.
  • DsbC fusion the level of expression for all target proteins is lower compared to Skp or MBP fusion.
  • MBP maltose binding protein
  • Skp fusions are found to produce more overall protein (ranging from 25-40% of the total protein), except for YopD.
  • Skp fusions are found to produce more soluble protein compared to MBP and DsbC fusion.
  • MBP seem to produce more soluble protein.
  • MBP molecular weight
  • the T7 protein kinase is used in accordance with the principles of the present invention to facilitate the expression and solubility of fusion partners. Since the carboxy terminal domain of the T7 protein kinase is toxic to E. coli (participating in the shut-off of host transcription), it is preferred to employ (as a fusion partner of the target protein) a fragment of the T7 protein kinase that lacks the toxic portion of the complete protein. Alternatively, the complete T7 protein may be used as a fusion molecule, preferably with one or more residues thereof mutated to eliminate or reduce the toxicity.
  • SEQ ID NO:33 is a polynucleotide encoding the complete phage T7 protein kinase (SEQ ID NO:34).
  • SEQ ID NO:35 is the preferred polynucleotide encoding a preferred amino terminal phage T7 protein
  • SEQ ID NO:35 atgagatcca acattaccga catcatgaac gctatcgacg caatcaaagc actgccaatc tgtgaacttg acaagcgtca aggtatgctt atcgacttac tggtcgagat ggtcaacagc gagacgtgtg atggcgagct aaccgaacta aatcaggcac ttgagcatca agattggtgg actaccttga agtgtctcac ggctgacgca gggttcaaga tgctcggtaa tggtcacttc tctt atagtcaccc gctgctacct aacagagtga ttaaggtggg cttaagaa gaggattcag gcgcaca
  • T-PK Bacteriophage T7 early gene 0.7 encodes two activities. About two-third of amino end of the protein encodes protein kinase activity, while the C- terminal one-third end is responsible for host transcription shutoff following infection (Brunoskis, 1. et al. (1977) "THE PROCESS OF INFECTION WITH BACTERIOPHAGE T7. VI. A PHAGE GENE CONTROLLING SHUTOFF OF HOST RNA SYNTHESIS," Virology 50:322-327; Rothman-Denes, L.B. et al. (1973) "A T7 GENE FUNCTION REQUIRED FOR SHUT-OFF OF HOST AND EARLY T7 TRANSCRIPTION," In Fox, CF.
  • T-PK protein Although full-length T-PK protein is toxic and cannot be expressed in E. coli, a polypeptide lacking the C-terminal end can be cloned in a plasmid and overexpressed at a very high level in a completely soluble form (Michalewicz, J. et al.
  • the fusion is prepared by fusing a polynucleotide sequence encoding the PK to a polynucleotide encoding the target protein.
  • the PK-encoding sequence will be fused to the amino terminus of the polynucleotide encoding the target protein.
  • a protease recognition site will be included between the PK and target protein encoding sequences so as to permit the cleavage of the chaperone protein from the target protein.
  • a preferred protease recognition site is the site recognized by the 27 kDa catalytic domain of the Nuclear Inclusion a (NIa) protein of the tobacco etch virus ("TEV protease").
  • TEV protease recognition sites are disclosed by Carrington, J. C. et al. (1988) ["A VIRAL CLEAVAGE SITE CASSETTE: IDENTIFICATION OF AMINO ACID SEQUENCES REQUIRED FOR TOBACCO ETCH VIRUS POLYPROTEIN PROCESSING" Proc. Natl. Acad. Sci. USA 85:3391 -3395]; Dougherty, W. et al. (1989) ["Molecular Genetic Analysis Of A Plant Virus Polyprotein Cleavage Site: A Model," Virology 171 :356-364]; Parks, T. et al.
  • TEV tobacco etch virus
  • a polynucleotide encoding an affinity tag is also included so as to pe ⁇ nit the facile recovery of the expressed fusion protein.
  • Figure 8 illustrates the configuration of preferred PK fusion proteins.
  • FIG. 9 shows the vector diagram of a preferred PK vector (pDest590-d-PK), and the ability of the vector to facilitate the soluble expression of Hifla, Folliculin, a Folliculin domain (FD) and ILl 3. All of these proteins are highly insoluble when expressed in E. coli.
  • T7 protein kinase fusion of these proteins improved solubility quite remarkably, except for Hifla, where approximately 10% solubility was obtained.
  • almost 100% solubility of the Folliculin domain, 90% solubility of Folliculin and 25% solubility of ILl 3 were obtained.
  • an affinity tag (“Strep-tag II”) (Sigma Genosys) was incorporated into the fusion. As shown in Figure 9, the level of expression and solubility are similar with or without the affinity tag.
  • One-step purification of the fusion protein was done using a Strep- tactin column matrix (IDA, Germany). A portion of soluble fraction (L) of the fusion protein was loaded onto the column (200 ⁇ l). The flow-through fraction (F) was collected. The column was washed with a five-column volume of wash buffer (supplied the vendor).
  • FIG. 10 shows the corresponding purification of a fusion of PK and the DEFG Folliculin Fragment using a Strep-tactin column matrix (IDA, Germany).
  • Figure 13 shows a comparison of the level of expression and solubility of Folliculin with Strepll-Skp or StrepII-PK fusion partners (Tev5 and TevlO Fusions).
  • Strep-tagll (IBA Incorporated, Germany) is the affinity purification tag and PK is the T7 kinase fusion tag.
  • the sequence of Strep-tagll is:
  • Tev-5 and Tev-10 describe the linker between the TEV protease site and the beginning of the protein.
  • the sequences of Tev-5 and Tev-10 are provided below (SEQ ID NO: 39 and SEQ ID NO: 40, respectively; the underlined residues are the TEV protease recognition site)
  • SEQ ID NO: 40 ENLYFQGSGA GGSGAG
  • TEV protease recognition site will result in the release of the fusion partner from the proteins of interest by TEV protease.
  • Skp was shown to be a better fusion partner than MBP (maltose binding protein) for both expression as well as solubility.
  • MBP maltose binding protein
  • Figure 13 indicates that T7 Protein Kinase fusion produced more total protein as well as soluble compared to Skp fusion.
  • T7 Protein Kinase appears to be the best "soluble expression tag" of the present invention.
  • Figure 14 shows the corresponding purification of Strep-tag Il-PK-Folliculin TevlO fusion protein. Purification was done as described above.
  • TEV protease cleavage was done as described above (see Figure 12). Interestingly, with spacer region installed between the TEV protease cleavage site and the fusion protein more efficient cleavage occurred. However, optimal amounts of TEV protease and reaction condition are desired in order to obtain complete removal of the fusion partner.
  • Figure 15 shows TEV protease digestion of a StreplI-PK-TEV10- Folliculin fusion molecule.

Abstract

L'invention concerne des procédés et des compositions permettant de produire des protéines recombinées, en particulier en quantités importantes. Cette invention est particulièrement indiquée pour la production de protéines qui sont insolubles ou très faiblement solubles dans des tampons aqueux. Elle concerne plus spécifiquement l'utilisation de molécules hybrides, la protéine souhaitée étant produite sous forme d'une molécule hybride liée à une protéine facilitant la solubilité, telle qu'une protéine chaperon ou la protéine kinase du bactériophage T7.
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US9261510B2 (en) * 2007-04-20 2016-02-16 Roche Diagnostics Operations, Inc. Detection of primary infections with pathogens
WO2012027930A1 (fr) * 2010-08-31 2012-03-08 上海交通大学 Vecteur d'expression soluble d'une protéine exogène, ses méthodes de préparation et d'application
WO2012106615A1 (fr) * 2011-02-03 2012-08-09 Xoma Technology Ltd. Procédés et matériaux pour améliorer l'expression d'une protéine fonctionnelle dans des bactéries
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EP3517125A1 (fr) * 2018-01-24 2019-07-31 Xuanwu Hospital of Capital Medical University Récepteur d'antigène chimérique pour une prolifération ciblée in vitro efficace et ses utilisations
US11001639B2 (en) * 2018-01-24 2021-05-11 Xuanwu Hospital Capital Medical University Chimeric antigen receptor for efficient selective proliferation in vitro and uses thereof
CN111733178A (zh) * 2020-07-13 2020-10-02 山西中医药大学 一种提高蒙古黄芪的病程相关蛋白的可溶性表达量的重组表达载体

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