WO2003004599A2 - Produits de recombinaison d'expression procaryotique, leurs procedes de production et d'utilisation dans l'expression de proteines de recombinaison dans des systemes d'expression procaryotique - Google Patents

Produits de recombinaison d'expression procaryotique, leurs procedes de production et d'utilisation dans l'expression de proteines de recombinaison dans des systemes d'expression procaryotique Download PDF

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WO2003004599A2
WO2003004599A2 PCT/IL2002/000540 IL0200540W WO03004599A2 WO 2003004599 A2 WO2003004599 A2 WO 2003004599A2 IL 0200540 W IL0200540 W IL 0200540W WO 03004599 A2 WO03004599 A2 WO 03004599A2
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tat
expression construct
protein
derived peptide
factor
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PCT/IL2002/000540
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WO2003004599A3 (fr
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Yoav Peleg
Zeev Pancer
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Insight Biopharmaceuticals Ltd.
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Publication of WO2003004599A3 publication Critical patent/WO2003004599A3/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
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates to novel prokaryotic expression constructs, methods of generating same and methods of using same for expression of recombinant proteins in prokaryotic expression systems.
  • the prokaryotic expression constructs of the invention induce periplasmic translocation of the expressed proteins and increase recombinant protein expression and bacterial cell mass, resulting in larger quantities of recombinant protein production and substantial simplification of their isolation and purification to homogeneity.
  • Heterologous protein expression has been a technique employed for the past 3 decades, with bacterial expression being recognized as an efficient means of eukaryotic recombinant protein production (Carbon, J., (1993) Genes, Replicators and Centromeres: The First Artificial Chromosomes, in The Early Days of Yeast Genetics. Cold Spring Harbor Laboratory Press pp. 375-390 and Baneyx F., (1999), Recombinant protein expression in Escherichia coli Current Opinion in Biotechnology 10: 411-421).
  • E. coli ideally suited as an expression system for many proteins: it is easy to genetically manipulate and it grows quickly in inexpensive media.
  • it is easy to genetically manipulate and it grows quickly in inexpensive media.
  • not every gene can be expressed efficiently in this organism (Makrides, "Strategies for Achieving High-Level Expression of Genes in Escherichia coli", Microbiological Reviews, 60(3): 512-538, 1996; Swartz, "Advances in Escherichia coli Production of Therapeutic Proteins", Curr. Opin. Biotechnol., 12(2): 195-201, 2001).
  • Large-scale protein expression in E. coli typically entails bacterial cell growth to high density followed by induction or derepression of the recombinant gene promoter. Tight regulation of the promoter is essential for the synthesis of proteins that may be detrimental to host cells (Makrides, "Strategies for Achieving High-Level Expression of Genes in Escherichia coli", Microbiological Reviews, 60(3):512-538, 1996; Swartz, "Advances in Escherichia coli Production of Therapeutic Proteins", Curr. Opin. Biotechnol., 12(2): 195-201, 2001).
  • Widely used induction systems that enable better regulation of specific promoters comprise: thermal induction (pL promoter), chemical induction (trp, tac, trc promoters) or nutritional induction (phoA promoter).
  • Proteins expressed in E. coli can be directed either to the cytoplasm or periplasm, or secreted to the extracellular medium (Makrides, "Strategies for Achieving High-Level Expression of Genes in Escherichia coli", Microbiological Reviews, 60(3):512-538, 1996, Swartz, "Advances in Escherichia coli Production of Therapeutic Proteins", Curr. Opin. Biotechnol., 12(2):195-201, 2001).
  • Protein transport to the bacterial periplasm is a complex and incompletely understood process.
  • the transport of protein through the inner membrane to the periplasmic space requires the inclusion of a signal peptide.
  • the presence of a signal peptide does not always ensure efficient protein translocation through the inner membrane.
  • Periplasmic protein expression and accumulation offers several advantages for heterologous protein production: (i) the oxidizing environment of the periplasm often facilitates the proper folding of proteins; (ii) due to the relatively small number of E.
  • the Human immunodeficiency virus type I trans activating regulatory protein is an 86-amino acid RNA binding protein involved in replication of the virus.
  • the TAT protein acts in concert with eukaryotic cellular proteins to greatly increase expression of viral genes.
  • the TAT protein increases viral gene expression by acting on the elongation step of transcription. This occurs through the interaction of TAT with a cellular protein kinase complex known as TAK (also known as cyclin Tl/P-TEFb).
  • TAT interacts directly with cyclin Tl and recruits the TAK complex to the viral promoter.
  • TAK hyperphosphorylates a region of RNA polymerase (RNAP) II known as the carboxyl-terminal domain (CTD) which is required for TAT transactivation. Phosphorylation of the CTD is thought to regulate the elongation activity of RNAP II. Therefore hyperphosphorylation of the CTD by TAK is believed to promote the formation of highly processive elongation complexes and provide an explanation for the molecular mechanism of the TAT transactivation function (Yang X, et al (1997) TAK, an HIV-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. Proc. Natl.
  • Lentivirus TAT proteins specifically associate with a cellular protein kinase, TAK, that phosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: a candidate for a TAT cofactor J. Virol. 69: 1612-1620).
  • the HIV1 TAT gene has two exons. TAT amino acids 1-72 are encoded by exon l, ⁇ nd amino acids 73-86 are encoded by exon 2.
  • the full- length TAT protein contains a basic region and a cysteine-rich region.
  • the basic region i.e., amino acids 49-57
  • the cysteine-rich region mediates the formation of metal-linked dimers in vitro (Frankel, A. D. et al, (1988) Science 240: 70-73; Frankel, A.
  • TAT basic region or derivatives thereof, such as, for example, a TAT peptide flanked by two Glycine residues
  • TAT peptide flanked by two Glycine residues Fawell et al, (1994) " TAT-mediated Delivery of Heterologous Proteins into Cells", PNAS, 91(2): 664-668,; Vives et al, (1997) "A truncated HIV-1 TAT Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus", J. Biol.
  • the references disclose heterologous protein production covalently attached to a segment of the TAT protein in E. coli, and subsequent delivery of this construct as a means of delivering TAT-conjugated proteins into eukaryotic cell cytoplasms or nucleii. Bacterial subcellular localization was not addressed, nor was there an attempt to address TAT-mediated effects on increased bacterial protein production. Finally, the ability of the TAT- containing constructs to undergo cleavage of the N-terminal TAT segment, resulting in properly folded, mature heterologously expressed protein is a unique application and novel development of the present invention alone. All the above references result in TAT-conjugated products being isolated from bacterial expression systems and used for delivery in eukaryotic systems.
  • incorporation of a bacterial signal sequence in frame with the TAT-derived peptide and protein-of-interest provided for cleavage of the signal sequence and the TAT-derived sequence, and provided a mature, properly folded and functional protein, readily isolatable and purifiable.
  • the signal sequence thus functioning as providing a bacterial protease cleavage recognition sequence for cleavage of the periplasmic targeting, or TAT- derived peptide, and the cleavage sequence itself. Once cleaved, the remaining protein-of-interest folds properly within the oxidizing environment provided by the bacterial periplasm.
  • the present invention relates to novel prokaryotic expression constructs, methods of generating same and methods of using same for expression of recombinant proteins in prokaryotic expression systems.
  • the prokaryotic expression constructs of the present invention encode for a TAT- derived polypeptide fused to a protein-of-interest, targeting the resulting recombinant fusion polypeptide to the periplasm. Incorporation of a bacterial signal sequence, or other bacterial protease cleavage recognition sequence, within the construct facilitates TAT-derived polypeptide cleavage, releasing the mature recombinant protein-of-interest.
  • the mature recombinant protein-of-interest can undergo proper folding, and can be isolated therefrom to homogeneity with relative ease, and minimum expense.
  • Non-bacterial protease cleavage recognition sequence can be used for post isolation processing of the fusion polypeptide into the mature recombinant protein-of-interest.
  • the prokaryotic expression constructs of the invention increase recombinant protein expression, periplasmic translocation of the expressed proteins and also increases bacterial cell mass.
  • the present invention provides processes and products for the efficient production of mature recombinant proteins-of-interest using prokaryotic expression systems.
  • the method and products provided herein are novel and/or beneficial in that they provide for (i) high levels of expression of a protein-of-interest in prokaryotic cells; (ii) proper folding of a protein-of interest once expressed; and/or (iii) ease of isolation of the proteins-of-interest from a bacterial periplasm. It is one object of the present invention to provide methods, expression constructs and kits for producing a protein-of-interest in, and purifying the protein-of-interest substantially, exclusively from a bacterial periplasm.
  • an assay of determining whether a TAT-derived peptide is an effective periplasmic targeting sequence comprises introducing into bacteria an expression construct encoding a fusion polypeptide comprising the TAT-derived peptide and a reporter protein, and determining to what extent the fusion polypeptide accumulates within the periplasm, thereby determining whether the TAT-derived peptide is an effective periplasmic targeting sequence.
  • the expression construct further harbors a reporter sequence for ease of identification of expression within a bacterial periplasm.
  • This assay can be used, by one ordinarily skilled in the art, to determine which of the homologous TAT-derived sequences from, for example, any of the viral species listed below, and/or modifications thereof is most efficient for the purpose of implementing the various methods, constructs and kits of the present invention which are further described hereinafter.
  • a method of producing a protein-of-interest in, and purifying the protein-of- interest from, bacteria comprises introducing into the bacteria an expression construct encoding a fusion polypeptide, which comprises a TAT- derived peptide, a signal sequence and the protein-of-interest.
  • the TAT- derived peptide serves for transport of the fusion polypeptide from the bacterial cytoplasm to the periplasm, and the signal sequence facilitates processing the fusion polypeptide to a mature protein, consisting essentially of the protein-of-interest and substantially lacking the TAT-derived peptide and signal sequence.
  • the mature protein is then substantially exclusively purified from the bacterial periplasm.
  • a method of producing a fusion polypeptide in, and purifying the fusion polypeptide from, bacteria comprises introducing into the bacteria an expression construct encoding the fusion polypeptide, which comprises a TAT-derived peptide, and a protein-of-interest.
  • the TAT-derived peptide serves for transport of the fusion polypeptide from the bacterial cytoplasm to the periplasm.
  • the fusion polypeptide is then substantially exclusively purified from the bacterial periplasm.
  • the fusion polypeptide further comprises a protease cleavage recognition sequence positioned between the TAT-derived peptide and the protein-of-interest
  • the method further comprises cleaving the fusion polypeptide with a protease specific to the protease cleavage recognition sequence, releasing the protein-of interest pre or post purification.
  • purifying the fusion polypeptide or the protein-of interest substantially exclusively from the bacterial periplasm comprises (a) isolating the bacterial periplasmic compartment from other subcellular compartments; (b) lysing the bacterial periplasmic compartment; and (c) purifying the protein of interest or fusion polypeptide.
  • isolating the bacterial periplasmic compartment utilizes the methods of subcellular fractionation, differential gradient centrifugation and/or gel electrophoresis.
  • purifying the fusion polypeptide or protein-of-interest utilizes methods of purification and analysis such as, but not limited to, column chromatography, electrophoresis, filtration, ultrafiltration, gradient centrifugation, HPLC, Western blot analysis, mass spectroscopy, GLC, and/or immunocytochemistry.
  • a prokaryotic cell engineered to express a fusion polypeptide comprising a TAT-derived peptide, a signal sequence and a protein-of-interest, wherein the TAT-derived peptide serves for transport of the fusion polypeptide to the periplasm of the prokaryotic cell and the signal sequence facilitates processing of the fusion polypeptide to yield a mature protein consisting essentially of the protein-of-interest and lacking the TAT-derived peptide and said signal sequence.
  • a prokaryotic cell engineered to express a fusion polypeptide comprising a TAT-derived peptide, a protease cleavage recognition sequence and a protein-of-interest, wherein the protease cleavage recognition sequence is positioned between the TAT-derived peptide and the protein-of-interest, whereby the TAT-derived peptide serves for transport of the fusion polypeptide to the periplasm of the prokaryotic cell.
  • nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide harboring an intact polylinker cloning sequence being operably linked to the first polynucleotide and a third polynucleotide harboring a prokaryotic promoter, being operably linked to the first polynucleotide.
  • the nucleic acid expression construct further comprises a polynucleotide encoding a protease cleavage recognition sequence in frame with the TAT-derived peptide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide encoding a signal sequence in frame with the TAT-derived peptide, and a third polynucleotide harboring a polylinker cloning sequence, being operably linked to the second polynucleotide and a fourth polynucleotide harboring a prokaryotic promoter, being operably linked to the first polynucleotide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide encoding a protease cleavage recognition sequence in frame with the TAT-derived peptide, a third polynucleotide encoding a protein-of-interest in frame with the protease cleavage recognition sequence and a fourth polynucleotide harboring a prokaryotic promoter, being operably linked to the first polynucleotide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide encoding a signal sequence in frame with the TAT-derived peptide, a third polynucleotide encoding a protein-of-interest in frame with the signal sequence and a fourth polynucleotide harboring a prokaryotic promoter, being operably linked to the first polynucleotide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide encoding a mammalian secreted protein-of-interest in frame with the TAT-derived peptide and a third polynucleotide harboring a reporter gene, being operably linked to the second polynucleotide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT-derived peptide, a second polynucleotide encoding a mammalian, non-nuclear, protein-of-interest in frame with the TAT-derived peptide, and a third polynucleotide harboring a reporter gene, being operably linked to the second polynucleotide.
  • kits comprising any of the above expression constructs and optionally reagents required for bacterial transformation and or transfection, including, for example, buffers, competent bacteria, phage packaging proteins, helper phages, enzymes, such as restriction endonucleases, ligases and DNA polymerases, and the like.
  • prokaryotic cell engineered to express the expression constructs described herein.
  • the prokaryotic cell utilized for expression of the fusion polypeptide, protein-of-interest and/or bacterial expression construct is a strain of a species selected from the group consisting of Escherichia, Streptococcus, Staphylococcus, Bacillus, Mycobacteria, Enterobacteriaceae, Vibrio, Campy lobacter, Helicobacter, Neisseria, Pseudomonas, Listeria, Francisella, Brucella, Legionella, Rickettsia, Coxiella, Haemophilus, Yersinia and
  • the TAT-derived peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 18), or alternatively, is derived from a virus selected from the group consisting of HIV 1, HIV-2, equine infectious anemia virus, simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), maedi- visna virus (MVV) and caprine arthritis-encephalitis-virus.
  • a virus selected from the group consisting of HIV 1, HIV-2, equine infectious anemia virus, simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), maedi- visna virus (MVV) and caprine arthritis-encephalitis-virus.
  • the TAT-derived peptide is N-terminal to the protein-of-interest in fusion polypeptides obtained from any of the above listed expression constructs encoding fusion polypeptides comprising a TAT- derived peptide and a protein-of-interest.
  • expression constructs containing polynucleotide sequences encoding the TAT-derived peptide are located upstream to sequences encoding the protein-of-interest.
  • the TAT-derived peptide is N-terminal to the signal sequence in fusion polypeptides obtained from any of the above listed expression constructs encoding fusion polypeptides comprising a TAT- derived peptide and a signal sequence.
  • expression constructs containing polynucleotide sequences encoding the TAT-derived peptide are located upstream to sequences encoding the signal sequence.
  • the signal sequence comprises a positively charged amino-terminus, a hydrophobic central region, and a neutral but polar carboxy-terminus.
  • the promoter sequence operably linked to the polynucleotides encoding the fusion polypeptide are either constitutive or inducible, and provide for low or high level expression of the construct.
  • the expression constructs contain a reporter gene, such as ⁇ -galactosidase, chloramphenicol acetyl transferase, luciferase and a fluorescent protein.
  • a reporter gene such as ⁇ -galactosidase, chloramphenicol acetyl transferase, luciferase and a fluorescent protein.
  • the expression constructs further comprise a polynucleotide encoding a positive or a negative selection marker.
  • the protein-of-interest may be selected from the group consisting of an insulin, an amylase, a protease, a lipase, a heparinase, a kinase, a phosphatase, a glycosyl transferase, a trypsinogen, a chymotrypsinogen, a carboxypeptidase, a hormone, a ribonuclease, a deoxyribonuclease, a triacylglycerol lipase, a phospholipase A2, an elastase, an amylase, a blood clotting factor, a UDP glucuronyl transferase, an ornithine transcarbamoylase, a cytochrome p450 enzyme, an adenosine deaminase, a serum thymic factor, a cytochrome p450 enzyme, an adenos
  • FIG. 1 is a schematic representation of the expression construct pTAT- STB-hGH. Relative positions of some functional and regulatory elements are shown in the diagram, including: Kan - Kanamycin-resistance gene; lad - lad repressor; ori - origin of replication; TAT-STB-hGH - TAT-derived sequence fused to E. coli heat stable enterotoxin II signal peptide and to human growth hormone.
  • FIG. 2A is a schematic representation of the expression cassette. Kpnl- Sad sites flank the expression cassette. Relative positions of some restriction sites, functional and regulatory elements are shown. Arrows indicate the direction of transcription from the heat-inducible pL promoter and from the constitutive promoter. Positioning of the multiple cloning site (MCS); and transcriptional termination (TT) site are as indicated.
  • FIG. 2B is a schematic representation of the expression construct hGHTSP/pACYC184. Relative positions of some functional and regulatory elements are as indicated, including: Tc - Tetracycline-resistance gene; ori- origin of replication; repressor - thermo-labile repressor; TSP-hGH - TAT- derived sequence fused to E. coli heat stable enterotoxin II signal peptide and to human growth hormone.
  • FIG. 3 is a photograph of a protein blot stained with coomasie blue revealing cytoplasmic accumulation of TAT-STB-hGH, and periplasmic accumulation of mature hGH, expressed in bacteria harboring constructs with the TAT-derived peptide. Large asterisks mark the non-processed fusion hGH and small asterisks mark the mature hGH.
  • Three different E. coli isolates harboring the pTAT-STB-hGH plasmid were examined, with and without induction, and sizes were compared to the protein molecular weight marker (Prestained Protein Marker, Broad Range, Cat. No.: P7708S, New England BioLabs), and presented in kilo-daltons (kDa).
  • FIG. 3 is a photograph of a protein blot stained with coomasie blue revealing cytoplasmic accumulation of TAT-STB-hGH, and periplasmic accumulation of mature hGH, expressed in bacteria harboring constructs with the TAT
  • FIG. 4 is a photograph of a protein blot probed with anti-hGH antibodies for the detection of recombinant hGH fusion polypeptides.
  • the upper panel shows positive staining for cytoplasmic and periplasmic proteins of 22-25 kDa in size.
  • the lower panel shows positive staining for proteins of 22-25 kDa in size, in samples of periplasmic proteins isolated before (-) and after (+) induction. Size differences reflect the periplasmic accumulation of mature hGH, as well as non-processed TAT-hGH, in respective samples.
  • FIG. 5 is a graph illustrating the growth rates of E. coli strains transformed with constructs with and without the TAT-derived sequence.
  • E. coli MM294 harboring expression constructs hGHSP/pACYC184 and hGHTSP/pACYC184, (without and with the TAT-derived sequence, respectively) were grown in a 5 liter fermenter. Samples were taken as indicated, for measurements of optical density (OD) at 600 nm. Open and closed circles represent growth of E. coli harboring expression construct hGHTSP/pACYC184 and hGHSP/pACYC184, respectively. Arrow indicates time of induction, when the culture reached 42 °C.
  • the present invention is of (i) novel prokaryotic expression constructs; (ii) methods of generating the expression constructs; and (iii) methods of using the expression constructs for the expression of recombinant proteins-of-interest in prokaryotic expression systems.
  • the prokaryotic expression constructs encode for fusion polypeptides which comprise a TAT-derived peptide, and optionally a prokaryotic signal sequence, or a protease cleavage recognition sequence, fused to a molecule of interest, resulting in the targeting of the recombinant fusion polypeptide to the bacterial periplasm, wherein optionally the TAT-derived peptide is cleaved and the remaining protein-of- interest undergoes proper folding, resulting in the production of a mature recombinant protein-of-interest.
  • the characterization of a novel periplasmic transport mechanism in prokaryotes for heterologously expressed, mature proteins-of-interest is described herein.
  • the transport mechanism utilizes the incorporation of a genetic fusion of a TAT-derived peptide and a protein-of-interest, to target the translated fusion polypeptide to the bacterial periplasm.
  • Incorporation of a bacterial signal sequence, functioning as a bacterial protease cleavage recognition sequence facilitates cleavage of the signal/cleavage sequence, as well as cleavage of the TAT-derived peptide.
  • TAT-derived peptide utilized in these studies comprises amino acids 47 to 57 of the TAT protein of HIV-1 (YGRKKRRQRRR) (SEQ ID 18). It is to be understood, and as is further defined hereinunder, that other sequences of any TAT protein may be similarly utilized to target fusion polypeptides containing a protein-of-interest to a bacterial periplasm.
  • modified sequences may include, for example, amino acids similar to those amino acids naturally present in the TAT protein (e.g., basic and hydrophylic amino acids), the absence of at least one amino acid and the addition of at least one amino acid.
  • TAT proteins have been identified in several viral species including, but not limited to, human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2), equine infectious anemia virus, simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), maedi-visna virus (MW) and caprine arthritis-encephalitis-virus.
  • TAT sequences which can be used in context of the present invention are described in Wender, PA (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: Peptoid molecular transporters. PNAS. 97(24)13003- 13008; and Futaki S (2001) Arginine-rich peptides. JBC 276(8)5836-5840.).
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 5 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 2 residues, although in some cases deletions may be larger.
  • substitutions may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of TAT derived peptide are desired, substitutions are generally made in accordance with the following chart:
  • TAT-derived peptides may be evaluated for their potential as periplasmic targeting sequences, and will be further discussed hereinbelow.
  • TAT-derived peptide was sufficient to direct heterologously expressed fusion polypeptides to the bacterial periplasm.
  • Western blots of human growth hormone (hGH) - TAT fusion polypeptides readily localized to bacterial periplasms.
  • Incorporation of a signal peptide, in this case the heat-stable enterotoxin II signal peptide (STB) further resulted in processing of the fusion polypeptide to yield the mature, appropriately sized (22 kDa) hormone, as opposed to the fusion peptide isolated by the TAT-hGH construct alone (24 kDa). Both constructs provided greater amounts of hGH as compared to constructs harboring STB-hGH alone, without inclusion of the TAT-derived peptide.
  • STB enterotoxin II signal peptide
  • the study presented herein is therefore the first to demonstrate that a viral derived protein product, TAT, directs protein translocation to the periplasmic space when the TAT-derived peptide is produced in frame with the protein-of-interest. Furthermore, the study presented herein is the first to describe the incorporation of bacterial signal sequence, in this case functioning as a bacterial protease recognition sequence, that facilitates cleavage of the TAT- derived peptide and signal sequence, releasing a mature recombinant protein- of-interest.
  • Translocation of the mature protein-of-interest within the periplasmic space facilitated proper protein folding and enabled protein function, and thus the present invention provides a novel and highly efficient means of high yield heterologous expression and ease of purification of a protein-of-interest in a bacterial cell.
  • the phrase "mature protein-of-interest” includes heterologously expressed proteins that are processed and folded to assume a final sequence and three- dimensional structure equal or similar to published accounts of the protein.
  • expression construct includes nucleic acid vectors that contain gene sequences essential for maintenance and propagation of the vector and directing/regulating the transcription/translation of inserted or subcloned sequences of interest.
  • fusion polypeptide and “fusion protein” may be used interchangeably and refer to a genetically engineered covalently linked protein derived from the joint, in- frame expression, of two or more heterologous nucleic acid sequences.
  • signal sequence refers to a short (e.g., 15-40) amino acid sequences, which allow proteins to transport through the bacterial inner membrane to the periplasm. During transport of proteins out of the cytoplasm, the signal peptide is typically removed by signal peptidases, thereby releasing a mature protein at the desired non-cytoplasmic location.
  • periplasm is understood to be the space between the bacterial inner and outer membranes.
  • TAT-derived peptide encompasses naturally appearing and man modified peptide sequences derived from any TAT protein of any virus whose genome encodes for a TAT protein and that is positively functional in the assay of determining whether a TAT-derived peptide is an effective periplasmic targeting sequence. Modifications to a naturally occurring TAT-derived peptide may include addition, deletion or substitution of one or more amino acids, as well known in the art. The periplasmic targeting activity of each such modified sequence can be determined via the assay described herein.
  • an assay of determining whether a TAT-derived peptide is an effective periplasmic targeting sequence comprises introducing into bacteria an expression construct encoding a fusion polypeptide comprising the TAT-derived peptide and a reporter protein and determining to what extent said fusion polypeptide accumulates within the periplasm, thereby determining whether the TAT-derived peptide is an effective periplasmic targeting sequence.
  • Experimental methods effective in implementing the above assay are described in the preferred embodiments and Examples sections of this application and/or are otherwise well known to the skilled artisan.
  • the assay according to this aspect of the present invention is useful in identifying novel, naturally occurring and/or man modified TAT- derived sequences, effective in periplasmic targeting of proteins fused thereto and hence useful in context of the various embodiments and aspects of the present invention.
  • a method of producing a protein-of-interest in, and purifying the protein-of- interest from, bacteria comprises introducing an expression construct encoding a fusion polypeptide into the bacteria.
  • the expression construct comprises a TAT-derived peptide, a signal sequence and the protein- of-interest, wherein the TAT-derived peptide serves for transporting the fusion polypeptide from the cytoplasm to the periplasm of the bacteria and the signal sequence facilitates processing of the fusion polypeptide to a mature protein.
  • the mature protein thus consists essentially of the protein-of-interest and substantially lacks the TAT-derived peptide and signal sequence.
  • the mature protein is then purified substantially exclusively from the bacterial periplasm.
  • an additional method of producing a protein-of-interest in, and purifying the protein-of-interest from, bacteria comprises introducing an expression construct encoding a fusion polypeptide into the bacteria.
  • the expression construct comprises a TAT-derived peptide, a protease cleavage recognition sequence and the protein-of-interest, wherein the TAT-derived peptide serves for transporting the fusion polypeptide from the cytoplasm to the periplasm of the bacteria.
  • the protease cleavage recognition sequence facilitates processing of the fusion polypeptide to a mature protein-of-interest by either by cleavage of the TAT-derived peptide and the protease cleavage recognition sequence by bacterial proteases prior to isolation from the periplasm, or by cleavage of the TAT-derived peptide and the protease cleavage recognition sequence by a protease post isolation from the periplasm.
  • the mature protein consists essentially of the protein-of-interest and substantially lacks the TAT-derived peptide and the protease cleavage recognition sequence.
  • a method of producing a fusion polypeptide in, and purifying a fusion polypeptide from, bacteria comprises introducing into the bacteria an expression construct encoding the fusion polypeptide comprising a TAT-derived peptide, and a protein-of-interest.
  • the TAT-derived peptide serves for transport of the fusion polypeptide from the cytoplasm to the periplasm of the bacteria. Once translocated to the bacterial periplasm, the fusion polypeptide is purified substantially exclusively from the bacterial periplasm.
  • TAT-derived peptide in the methods disclosed herein of construction and isolation of fusion polypeptides containing a protein-of- interest, and fusion polypeptides containing a protein-of-interest and signal sequence or a protease cleavage sequences, processed and ultimately lacking the signal sequence and the TAT-derived peptide, facilitates increased yield of the protein-of-interest.
  • purification of fusion polypeptides and proteins-of- interest from the bacterial periplasm may be accomplished by isolating the bacterial periplasmic compartment from other subcellular compartments, lysing the bacterial periplasmic compartment and purifying the protein of interest from the lysate, using, for example, at least one of the following purification and/or analysis methods: column chromatography, electrophoresis, filtration, ultrafiltration, gradient centrifugation, preparative HPLC, analytic HPLC, Western blot analysis, mass spectroscopy, GLC, and/or immunocytochemistry.
  • polynucleotide segments encoding a TAT-derived peptide, the protein-of-interest and optionally a signal sequence or a protease cleavage recognition sequence can be ligated into commercially available expression construct systems suitable for transforming bacterial cells and for directing the expression of the fusion polypeptide within the transformed cells.
  • Suitable bacterial expression constructs for use with the present invention include, but are not limited to the pCAL, pUC, pET, pETBlueTM (Novagen), pBAD, pLEX, pTrcHis2, pSE280, pSE380, pSE420 (Invitrogen), pKK223-2 (Clontech), pTrc99A, pKK223-3, pRIT2T, pMC1871, pEZZ 18 (Pharmacia), pBluescript II SK (Stratagene), pALTER-Exl, pALTER-Ex2, pGEMEX (Promega), pFivE (MBI), pQE (Qiagen) commercially available expression constructs, and their derivatives.
  • the construct may also include, a plasmid, a bacmid, a phagemid, a cosmid, or a bacteriophage.
  • Bi-functional or shuttle vectors suitable for propagation and gene expression both in prokaryote and eukaryote organisms are also within the scope of the present invention.
  • Nucleotide sequences are typically operably linked to, i.e., positioned, to ensure the functioning of an expression control sequence.
  • These expression constructs are typically replicable in the cells either as episomes or as an integral part of the cell's chromosomal DNA, and may contain appropriate origins of replication for the respective prokaryotic strain employed for expression.
  • expression constructs contain selection markers, such as for example, tetracycline resistance, ampicillin resistance, kanamycin resistance or chlormaphenicol resistance, facilitating detection and/or selection of those bacterial cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Pat. No. 4,704,362). These markers, however, are not exclusionary, and numerous others may be employed, as known to those skilled in the art. Indeed, in a preferred embodiment of the present invention expression constructs contain both positive and negative selection markers.
  • reporter genes may be incorporated within expression constructs to facilitate identification of transcribed products. Accordingly, in a preferred embodiment of the present invention, reporter genes utilized are selected from the group consisting of ⁇ -galactosidase, chloramphenicol acetyl transferase, luciferase and a fluorescent protein, e.g., green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Prokaryotic promoter sequences regulate expression of the encoded polynucleotide sequences, and in preferred embodiments of the present invention, are operably linked to polynucleotides encoding the TAT-derived peptide and polynucleotides encoding the protein-of-interest. In additional preferred embodiments of the present invention, these promoters are either constitutive or inducible, and provide a means of high and low levels of expression of the fusion polypeptides.
  • promoters including the T7 promoter system, the lactose promoter system, typtophan (Trp) promoter system, Trc/Tac promoter systems, beta-lactamase promoter system, tetA promoter systems, arabinose regulated promoter system, Phage T5 promoter, or a promoter system from phage lambda, may be employed, and others, as well, all comprise preferred embodiments of the present invention.
  • the promoters will typically control expression, optionally with an operator sequence and may include ribosome binding site sequences for example, for initiating and completing transcription and translation.
  • the vector may also contain expression control sequences, enhancers that may regulate the transcriptional activity of the promoter, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter and other necessary information processing sites, such as RNA splice sites, polyadenylation sites and transcription termination sequences as well as any other sequence which may facilitate the expression of the inserted nucleic acid.
  • premature proteins For proteins normally expressed within a bacterial cell, protein export from the cytoplasm to the periplasmic space as premature forms naturally occurs. These premature proteins have short (15-30) specific amino acid sequences, which allow proteins to transport through the inner membrane to the periplasm. These short amino acid sequences are commonly referred to as signal peptides.
  • the premature protein interacts with the bacterial secretion apparatus, and during transport of the proteins out of the cytoplasm, the signal peptide is typically removed by signal peptidase thereby leaving a mature protein at the desired non-cytoplasmic location.
  • the features of a typical bacterial signal peptide include a positively charged amino-terminus (n- region), a hydrophobic central region (h-region), and a neutral but polar carboxy-terminus (c- region).
  • a helix-breaking residue usually marks the boundary between the h-region and the c-region at the -6 (P6) position relative to the cleavage site.
  • the cleavage recognition sequence consists of small residues at the -1 (PI) and -3 (P3) positions relative to the cleavage site (Edman, M., Jarhede, T., Sjostrom, M., and Wieslander, A. (1999) Different sequence patterns in signal peptides in signal peptidases from mycoplasmas, other gram- positive bacteria, and Escherichia coli: multivariate data analysis. Proteins Struc. Func. Gen. 35: 195-205; Jones, J. D., and Giersch, L. M.
  • a prokaryotic nucleic acid expression construct comprising a first polynucleotide encoding a fusion polypeptide comprising a signal sequence, TAT-derived peptide and a protein-of-interest.
  • the TAT-derived peptide serves for transport of the fusion polypeptide to the periplasm of the bacterial cell expressing the construct, and expression of the signal sequence facilitates cleavage of both the TAT-derived peptide and signal sequence, resulting in a mature protein-of-interest, devoid of the TAT-derived peptide and signal sequence.
  • the construct also comprises a second polynucleotide harboring a prokaryotic promoter, being operably linked to the first polynucleotide, providing a means for regulating expression of the construct.
  • the construct additionally comprises a reporter gene and positive and/or negative selection markers.
  • the signal sequence functions to cleave both the signal sequence itself, and the TAT-derived peptide from the protein-of-interest, it is apparent that the signal sequence is functioning as a bacterial protease cleavage recognition sequence.
  • a nucleic acid expression construct comprising a polynucleotide encoding a TAT-derived peptide, a polynucleotide encoding a protease cleavage recognition sequence in frame with the TAT-derived peptide, a polynucleotide encoding a protein-of-interest in frame with the protease cleavage recognition sequence and a polynucleotide harboring a prokaryotic promoter, being operably linked to the polynucleotide encoding the TAT-derived peptide, providing a means for regulating expression of the construct.
  • the construct similarly additionally comprises a reporter gene and positive and/or negative selection markers.
  • the present invention is the first demonstration of a TAT- derived peptide functioning as a periplasmic targeting sequence.
  • Purification of fusion polypeptides of heterologously expressed proteins-of-interest from bacterial cells often result in poor yields, improperly folded or unfolded proteins, and proteins improperly processed, or impossible to purify from bacterial cell cytoplasms.
  • the present invention circumvents these difficulties by providing a means to direct these heterologous proteins-of-interest to the periplasmic space, where proper folding often occurs as a function of the oxidizing environment of the intracellular compartment. Isolation and purification of the protein is more readily accomplished as few bacterial proteins are located within the periplasmic space.
  • TAT-derived peptide resulted in markedly higher production of the protein-of-interest following induction, and hence provides a superior means for isolation and purification of fusion-polypeptides containing a protein-of-interest or the protein-of-interest itself in a mature form.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT- derived peptide, a second polynucleotide harboring an intact polylinker cloning sequence operably linked to the TAT-derived peptide and a third polynucleotide harboring a prokaryotic promoter, operably linked to the TAT- derived peptide.
  • This expression construct provides a means therefore of producing a fusion polypeptide containing a protein-of-interest subcloned within the polylinker cloning sequence, that when expressed will be targeted to the bacterial periplasm.
  • a reporter gene may be included in the construct as well, as are positive and/or negative selection markers.
  • the phrase "intact polylinker cloning sequence” refers to a non-interrupted polylinker cloning sequence which includes at least 50, 60, 70 or at least 80 nucleotides and harbors at least 2, preferably, at least 5, at least 10, at least 15 or at least 20 unique restriction endonuclease recognition sequences, some of which may be overlapping in sequence, at least one of which, preferably 2-10 of which are recognized by 6 cutter restriction endonucleases and at least one is recognized by a 8 cutter restriction endonuclease.
  • mammalian secreted proteins-of-interest may be produced and targeted to the bacterial periplasm by incorporation within a bacterial expression construct encoding a TAT-derived peptide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT- derived peptide, a second polynucleotide encoding a mammalian secreted protein-of-interest in frame with the TAT-derived peptide, and a third polynucleotide harboring a reporter gene, being operably linked to said second polynucleotide.
  • bacterial protease recognition sequence may be encoded as well, in order to provide a means of expressing a mature and processed mammalian secreted protein-of- interest.
  • Positive and negative selection markers may be incorporated as indicated above.
  • non-nuclear mammalian proteins-of-interest may be produced and targeted to the bacterial periplasm by incorporation within a bacterial expression construct encoding a TAT-derived peptide.
  • a nucleic acid expression construct comprising a first polynucleotide encoding a TAT- derived peptide, a second polynucleotide encoding a non-nuclear mammalian protein-of-interest in frame with the TAT-derived peptide, and a third polynucleotide harboring a reporter gene, being operably linked to said second polynucleotide.
  • bacterial protease recognition sequence may be encoded, in order to provide a means of expressing a mature and processed mammalian non-nuclear protein- of-interest. Positive and negative selection markers may be incorporated as well.
  • the TAT-derived peptide in the expression constructs described hereinabove, is N-terminal to the protein- of-interest, when expressed. In still other preferred embodiments, in the expression constructs described hereinabove, the TAT-derived peptide is N-terminal to the signal sequence.
  • the present invention provides for prokaryotic cells containing the constructs described herein and designed for expression of a protein-of-interest.
  • a prokaryotic cell engineered to express a fusion polypeptide comprising a TAT-derived peptide, a signal sequence and a protein-of-interest, wherein the TAT-derived peptide serves for transport of the fusion polypeptide to a periplasm of the prokaryotic cell and the signal sequence facilitates processing of the fusion polypeptide to yield a mature protein consisting essentially of the protein-of-interest and lacking the TAT-derived peptide and signal sequence.
  • a prokaryotic cell engineered to express a fusion polypeptide comprising a TAT-derived peptide, a protease cleavage recognition sequence and a protein-of-interest, wherein the protease cleavage recognition sequence is positioned between the TAT-derived peptide and protein-of-interest, whereby the TAT-derived peptide serves for transport of the fusion polypeptide to a periplasm of the prokaryotic cell.
  • references to a protein-of-interest may include proteins, such as, but not limited to, an insulin, an amylase, a protease, a lipase, a heparinase, a kinase, a phosphatase, a glycosyl transferase, a trypsinogen, a chymotrypsinogen, a carboxypeptidase, a hormone, a ribonuclease, a deoxyribonuclease, a triacylglycerol lipase, a phospholipase A2, an elastase, an amylase, a blood clotting factor, a UDP glucuronyl transferase, an ornithine transcarbamoylase, a cytochro
  • Prokaryotic cells are thus utilized for expressing any and all of the expression constructs listed herein, and are a means of producing the recombinant proteins-of-interest.
  • the prokaryotic cells utilized in any of these applications are of a strain of a species selected from the group consisting of Escherichia, Streptococcus, Staphylococcus, Bacillus, Mycobacteria, Enterobacteriaceae, Vibrio, Campylobacter, Helicobacter, Neisseria, Pseudomonas, Listeria, Francisella, Brucella, Legionella, Rickettsia, Coxiella, Haemophilus, Yersinia and Mycoplasma.
  • the expression construct will contain an appropriate origin-of-replication sequence for the respective prokaryotic strain employed for expression, selection markers and sequences encoding reporter polypeptides, and promoter or enhancer sequences operably linked to ensure the functioning of an expression control sequence.
  • the expression constructs, bacterial strains and conditions for introducing the constructs can be optimized empirically and hence provide a readily accessible system for heterologous expression of proteins-of-interest in prokaryotes.
  • kits comprising any of the expression constructs described herein and optionally enzymes, substrates and/or reagents for expression, verification and utilization of the expression constructs of the invention.
  • the kit further comprises cells into which the expression construct can be transformed or transfected.
  • the kit further comprises cells competent for genetic manipulation.
  • the kit further comprises oligonucleotide primers for the amplification, purification and subcloning of specific sequences of interest within the polylinker cloning sequence within the prokaryotic expression construct.
  • a 576 bp fragment of the mature human Growth Hormone cDNA (SEQ ID NO: 1) was amplified from Human Pituitary Gland cDNA (Clonetech, Cat. No. 7173-1) by PCR using the specific sense primer: hGH.F - 5'- GGGCTATGCATTCCCAACCA TTCCGTTATCCAGGC-3 ' (SEQ ID NO: 2) and a specific antisense primer: hGH.R- 5'-
  • PCR conditions were: denaturation at 94 °C for 2 minutes, addition of heat stable DNA polymerase and additional denaturation at 94 °C for 40 seconds followed by annealing at 60 °C for 80 seconds. 35 cycles of the following steps were then carried out: elongation at 72 °C for 3 minutes; denaturation at 94 °C for 40 seconds; and annealing at 60 °C for 80 seconds. The last step in the amplification was elongation at 72 °C for 5 minutes.
  • the hGH.F primer introduced a Nsil site and an in frame ATG codon.
  • the hGH.R primer introduced a Bam ⁇ l site and a translation stop codon.
  • the sequence of the PCR product was confirmed with sequence specific primers, using an automated DNA sequencer (Applied Biosy stems, Model 373A). Construction of IPTG-induced expression constructs: An expression construct, harboring the E. coli heat-stable enterotoxin II signal peptide (Picken et al, "Nucleotide Sequence of the Gene for Heat-Stable Enterotoxin II of Escherichia coli", Infect. Immun.
  • the DNA fragment encoded for the heat-stable enterotoxin STI signal peptide (designated as STB) was constructed by annealing oligonucleotides STB.F: TATGAAAAAG AATATCGCAT TTCTTCTTGC ATCTATGTTC GTTTTTTCTA TTGCTACAAA TGCCTATGCA TG (SEQ ID NO: 4) and STB.R: GATCCATGCA TAGGCATTTG TAGCAATAGA AAAAACGAAC ATAGATGCAA GAAGAAATGC GATATTCTTT TTCA (SEQ ID NO: 5).
  • the resulting double-stranded DNA fragment has a compatible Ndel overhang at the 5' end and an N overhang at the 3 'end that was generated following digestion with Nsil endonuclease.
  • the PCR product of the mature hGH cD ⁇ A was then digested with Nsil and BamHl.
  • a 3-piece ligation of the STB and the mature hGH cD ⁇ A into the Ndel and BamHl sites in the pET-24b plasmid ( ⁇ ovagen) resulted in the establishment of an expression construct, designated pSTB-hGH.
  • This expression construct encoded an STB-hGH open reading frame of 214 amino acids (SEQ ID NO: 6).
  • a second expression construct, containing the TAT-derived sequence amino terminal to the mature hGH was constructed as follows: The TAT- derived sequence was generated by annealing of two oligonucleotides mTATl .F: CATATGAAAG GCTATGGCCG CAAAAAACGT CGCCAGCGTC GCCGTGGTGC A (SEQ ID NO: 7) and mTATl.R: CCACGGCGAC GCTGGCGACG TTTTGCGG CCATAGCCTT TCATATG (SEQ ID NO: 8). This resulted in generation of the mTATl sequence with an Ndel overhang at the 5' end and Nsil site at the 3' end.
  • the mTATl was then ligated with mature hGH removed from the plasmid pSTB- hGH with Nsil-BamRl endonuclease.
  • the ligation product was PCR amplified with a sense primer mTAThGH.F: AACATATGAA AGGCTATGGC CGCAA (SEQ ID NO: 9) and an antisense primer mTAThGH.R: AAAGGATCCA TTAGAAGCCA CAGCTGCCCT C (SEQ ID NO: 10)
  • the PCR product was digested with Ndel and BamHl endonucleases and ligated into the corresponding sites in the pET-24b plasmid (Novagen).
  • mTAT2.F CATATGAAAG GCTATGGCCG CAAAAAACGT CGCCAGCGTC GCCGTGGCGC A (SEQ ID NO: 12) and mTAT2.R: CCACGGCGAC GCTGGCGACG TTTTTTGCGG CCATAGCCTT TCATATG (SEQ ID NO: 13) were annealed, generating the mTAT2 sequence with an Ndel overhang at the 5' end and at the 3' end a site compatible for ligation with the 5' of cSTB fragment.
  • the cSTB fragment was assembled by annealing oligonucleotides cSTB.F: TTTCTTCTTG CATCTATGTT CGTTTTTTCT ATTGCTACAA ATGCCTATGC A (SEQ ID NO: 14) and cSTB.R: TAGGCATTTG TAGCAATAGA AAAAACGAAC ATAGATGCAA GAAGAAATGC G (SEQ ID NO: 15).
  • the cSTB fragment has a 5' end compatible for ligation with the 3' end of mTAT2 and 3' end, which is Nsil compatible.
  • the mTAT2 and cSTB fragments were then ligated to an Nyz ' I- BamHl fragment of the mature hGH cD ⁇ A obtained from plasmid pSTB- hGH.
  • the ligation product was PCR amplified with a sense primer mTAThGH.F (SEQ ID NO: 9) and antisense primer mTAThGH.R (SEQ ID NO: 10).
  • the PCR product was digested with Ndel and BamHl endonudeases and ligated into the corresponding sites in pET24b (Novagen).
  • the resulting expression construct designated pTAT-STB-hGH, encoded a TAT-STB-hGH open reading frame of 224 amino acids (SEQ ID NO: 16).
  • a schematic representation of the expression construct pTAT-STB-hGH is shown in Figure 1.
  • Construction of a heat-inducible expression construct Construction of the heat inducible expression constructs hGHSP/pACYC184 and hGHTSP/pACYC184 was a multiple step process.
  • An expression cassette containing the following components was assembled ( Figure 2a): ⁇ gtl l fhermo-labile repressor under the control of a synthetic constitutive promoter; the phase ⁇ pL promoter; a multiple cloning site (MCS); and a transcriptional termination site (TT). Transcription from the constitutive promoter controlling the ⁇ gtl 1 thermo-labile repressor is in opposite direction to transcription from the ⁇ pL promoter.
  • the entire expression cassette including the regulatory elements and the hGH sequence were removed from vectors hGHTSP/pVlRepl and hGHSP/pVlRepl by Sacl-Kpnl digestion.
  • the Sacl-Kpnl fragments were blunt-ended using T7 DNA polymerase and ligated to a blunt-ended Ahdl- Xmnl fragment from the plasmid pACYC184 (New-England BioLabs, GeneBank Accession No. X06403).
  • FIG. 2B Schematic representation of the expression construct hGHTSP/pAC YC 184 is shown in Figure 2B .
  • Protein expression using an IPTG induction system The three constructs pSTB-hGH, pTAT-hGH and pTAT-STB-hGH, were used to transform competent cells of E. coli strain BL21(DE3) (Stratagene). Single colonies were selected. Bacteria, harboring these plasmids, were grown in medium containing 10 grams/liter (g/L) tryptone, 5 g/L yeast extract and 10 g/L sodium chloride supplemented with 30 ⁇ g/ml of kanamycin (LB-kan). Following overnight growth, cultures were backdiluted
  • the bacteria was subjected to osmotic shock by incubation with a solution containing 20 mM Tris-HCl at pH 8, 2.5 mM EDTA (ethylenediaminetetraacetic acid, pH 8.0) and 20 % (w/v) sucrose (osmotic shock solution No. 1), and incubated for 10 minutes at 4 °C. Following centrifugation (18,500 g, 5 minutes) the cell pellet was resuspended in the same volume as above with a solution containing 20 mM Tris-HCl, pH 8.0 and 2.5 mM EDTA (pH 8.0) (osmotic shock solution No. 2) and incubated for 10 minutes at 4 °C.
  • TAT-derived sequences inserted into a periplasmic-targeting signal peptide enabled the transport of the fusion polypeptide to the periplasm, correct processing of the synthetic signal sequence, and enhanced the level of accumulation of the mature polypeptide in the periplasm.
  • the heat inducible expression construct hGHTSP/pACYC184 was used to transform E. coli strain MM294 (ATCC 33625) establishing the hGH expression clone. Transformation of the expression construct to E. coli was carried out by electroporation using the BioRad Micro Pulser Electroporator (Cat. No. 165-2100). Transformation was performed according to the manufacturer recommendations. Transformants were plated on LB agar plates supplemented with 12 ⁇ g/ml tetracycline, and incubated overnight at 30 °C.
  • a single colony was inoculated into broth containing 5 ml LB-tet medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L sodium chloride supplemented with 12 ⁇ g/ml of tetracycline) and incubated at 30 °C for roughly 14 hours.
  • the culture was then backdiluted to an OD of 0.1 (600 nm) in fresh LB medium (without tetracycline).
  • the bacterial culture was grown at 30 °C for 2 hours and protein expression was induced by elevation of the culture temperature to 42 °C for 6 hours.
  • Cells were harvested and periplasmic proteins were extracted using the following method: Cells were brought to an OD of 5 at 600 nm.
  • Osmotic shock was induced with osmotic shock solution No. 1 (detailed in Example 1 above). Cells were incubated on ice for 10 minutes followed by additional centrifugation (18,500 g, 5 minutes). The cell pellet was resuspended in the same volume as above with osmotic shock solution No. 2 (detailed in Example 1). Cells were then incubated on ice for 10 minutes followed by centrifugation (18,500 g, 5 minutes). The supernatant obtained (periplasmic fraction) was concentrated using theYM-10 Centricon (Millipore, Cat. No. 4321), following manufacturer's instructions and kept overnight at 4 °C.
  • One ml of the above concentrate was acidified by the addition of 15 ⁇ l glacial acetic acid.
  • the acidified concentrate was filtered through a 0.45 micron PVDF syringe driven filter unit (Millex-HV, Millipore, Cat. No.: SLHV R04 NL) and 250 - 900 ⁇ l were injected on a Waters Delta Prep HPLC system (Delta Prep 4000, Preparative Chromatography System) fitted with a Vydac C4, 300 A, reverse phase column (4.6 x 250 mm).
  • the mobile phase was: A - 0.1 % trifluoroacetic acid in water, B - 0.1% trifluoroacetic acid in acetonitrile.
  • the flow rate was 1 ml/minute.
  • the gradient used was 20-80 % of B in 60 minutes. Detection was done at 280 nm.
  • the standard (Genotropin, Pharmacia-Upjohn, Sweden) eluted under these conditions with a retention time of 24.5 minutes.
  • the initial fermentation medium contained the following ingredients: 21.9 mM potassium dibasic phosphate, 13.9 mM sodium monobasic phosphate, 29.6 mM potassium chloride, 55.7 mM ammonium sulfate, 5 mM sodium citrate, 14.7 mM magnesium sulfate, 1.11 % tryptone, 1.11 % yeast extract, 0.11 % glucose, 0.002 % ferric sulfate, 0.4 ml/L of antifoam and 0.5 mg/L tetracycline.
  • a trace element solution (3.7 ml/5 L) was added, containing 100 mM ferric sulfate and 30 mM of each of the following: zinc sulfate, cobalt chloride, sodium molybdate, copper sulfate, boric acid and manganese sulfate.
  • a pH of 7.2 was maintained throughout the process by the addition of H 2 S0 solution and ammonium hydroxide.
  • 50 % (w/v) of a glucose solution was fed to the fermenter from initiation of the process. Adjustment of glucose feeding was done during the process in order to maintain a glucose level of below 4 g/L.
  • Dissolved oxygen was measured by an on-line oxygen electrode and was set to 30%. The dissolved oxygen setting was maintained by increasing the agitation, airflow and oxygen supplementation during the process.
  • Cell growth was performed at 30 °C for 5.5 hours, followed by increasing the culture temperature to 42° C over a period of 30 minutes.
  • Induction of hGH expression was performed at 42° C for 6 hours. Bacterial cells were harvested via centrifugation (6,200 g, 15 minutes at 4°C) and the cell pellet was stored at -20° C.

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Abstract

L'invention porte sur un procédé de production d'un polypeptide de fusion polypeptide dans des bactéries et sur son procédé de purification à partir des bactéries. Le procédé consiste à introduire dans les bactéries un produit de recombinaison d'expression codant le polypeptide de fusion qui comprend un peptide dérivé de TAT et une protéine d'intérêt. Le polypeptide dérivé de TAT sert au transport du polypeptide de fusion du cytoplasme bactérien au périplasme. Le polypeptide de fusion est ensuite purifié exclusivement à partir du périplasme bactérien.
PCT/IL2002/000540 2001-07-02 2002-07-02 Produits de recombinaison d'expression procaryotique, leurs procedes de production et d'utilisation dans l'expression de proteines de recombinaison dans des systemes d'expression procaryotique WO2003004599A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2005019466A3 (fr) * 2003-08-20 2005-05-06 Sandoz Ag Procede de preparation de polypeptides recombinants
WO2005017174A3 (fr) * 2003-08-13 2005-08-18 Sandoz Ag Procédé de purification de polypeptides recombinés
JP2007501623A (ja) * 2003-08-13 2007-02-01 サンド・アクチエンゲゼルシヤフト 組換えポリペプチドの生産のための発現ベクター、形質転換宿主細胞及び発酵方法
CN102766645A (zh) * 2012-06-08 2012-11-07 武汉凯肽来生物科技有限公司 一种促进黄鳝生长的口服重组蛋白tat-gh及制备方法和应用
WO2014084432A1 (fr) * 2012-11-30 2014-06-05 경상대학교 산학협력단 Vecteurs d'expression d'helicobacter pylori
CN111705032A (zh) * 2020-07-09 2020-09-25 科赫生物科技（北京）有限公司 一种原核表达细胞及其制备方法和应用

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WO2005017174A3 (fr) * 2003-08-13 2005-08-18 Sandoz Ag Procédé de purification de polypeptides recombinés
JP2007501623A (ja) * 2003-08-13 2007-02-01 サンド・アクチエンゲゼルシヤフト 組換えポリペプチドの生産のための発現ベクター、形質転換宿主細胞及び発酵方法
US7785830B2 (en) 2003-08-13 2010-08-31 Sandoz Ag Expression vectors, transformed host cells and fermentation process for the production of recombinant polypeptides
WO2005019466A3 (fr) * 2003-08-20 2005-05-06 Sandoz Ag Procede de preparation de polypeptides recombinants
JP2007502609A (ja) * 2003-08-20 2007-02-15 サンド・アクチエンゲゼルシヤフト 組換えポリペプチドを調製するための方法
EP2302068A1 (fr) * 2003-08-20 2011-03-30 Sandoz AG Procédé de préparation de polypeptides recombinants
JP4750030B2 (ja) * 2003-08-20 2011-08-17 サンド・アクチエンゲゼルシヤフト 組換えポリペプチドを調製するための方法
CN102766645A (zh) * 2012-06-08 2012-11-07 武汉凯肽来生物科技有限公司 一种促进黄鳝生长的口服重组蛋白tat-gh及制备方法和应用
WO2014084432A1 (fr) * 2012-11-30 2014-06-05 경상대학교 산학협력단 Vecteurs d'expression d'helicobacter pylori
CN111705032A (zh) * 2020-07-09 2020-09-25 科赫生物科技（北京）有限公司 一种原核表达细胞及其制备方法和应用

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