WO2006067511A1 - Gene expression technique - Google Patents

Gene expression technique Download PDF

Info

Publication number
WO2006067511A1
WO2006067511A1 PCT/GB2005/005085 GB2005005085W WO2006067511A1 WO 2006067511 A1 WO2006067511 A1 WO 2006067511A1 GB 2005005085 W GB2005005085 W GB 2005005085W WO 2006067511 A1 WO2006067511 A1 WO 2006067511A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
plasmid
host cell
chaperone
sequence
Prior art date
Application number
PCT/GB2005/005085
Other languages
French (fr)
Inventor
Christopher John Arthur Finnis
Darrell Sleep
Gillian Shuttleworth
Original Assignee
Novozymes Delta Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/GB2004/005435 external-priority patent/WO2005061719A1/en
Priority claimed from PCT/GB2004/005462 external-priority patent/WO2005061718A1/en
Application filed by Novozymes Delta Limited filed Critical Novozymes Delta Limited
Priority to CN200580048615.0A priority Critical patent/CN101128587B/en
Priority to EP05823505.2A priority patent/EP1831375B1/en
Priority to JP2007547668A priority patent/JP5631533B2/en
Priority to AU2005317828A priority patent/AU2005317828A1/en
Priority to US11/722,539 priority patent/US9057061B2/en
Publication of WO2006067511A1 publication Critical patent/WO2006067511A1/en
Priority to US13/474,313 priority patent/US8969064B2/en
Priority to US14/605,314 priority patent/US20160186192A1/en

Links

Classifications

    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present application relates to gene expression techniques.
  • chaperones The class of proteins known as chaperones has been defined by Hard (1996, Nature, 381, 571-580) as a protein that binds to and stabilises an otherwise unstable conformer of another protein and, by controlled binding and release, facilitates its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation.
  • BiP also known as GRP78, Ig heavy chain binding protein and Kar2p in yeast
  • GRP78 Ig heavy chain binding protein and Kar2p in yeast
  • ER endoplasmic reticulum
  • Protein disulphide isomerase is a chaperone protein, resident in the ER that is involved in the catalysis of disulphide bond formation during the post- translational processing of proteins.
  • BiP may play a dual role in solubilising folding precursors and preventing the transport of unfolded and unassembled proteins.
  • WO 93/25676 taught that the two genes could be chromosomally integrated; one could be chromosomally integrated and one present on a plasmid; each gene could be introduced on a different plasmid; or both genes could be introduced on the same plasmid.
  • WO 93/25676 exemplified expression of antistasin from the plasmid pKH4 ⁇ 2 in yeast strains having a chromosomally integrated additional copy of a PDI gene (Examples 16 and 17); expression of antistasin from the vector K991 with an additional PDI gene copy being present on a multicopy yeast shuttle vector named YEp24 (Botstein et al, 1979, Gene, 8, 17-24) (Example 20); and expression of both the antistasin and the PDI genes from the yeast shuttle vector pCl/1 (Rosenberg et al, 1984. Nature, 312, 77-80) under control of the GALlO and GALl promoters, respectively.
  • Hayano et al, 1995, op. cit. described the introduction of genes for human lysozyme and PDI into a yeast host each on a separate linearised integration vector, thereby to bring about chromosomal integration.
  • bovine pancreatic trypsin inhibitor BPTI
  • the 2 ⁇ m-based expression plasmid was said to be counter-productive for the production of heterologous secreted protein.
  • Bao et al concluded that over-expression of the KlPDIl gene was toxic to K. lactis cells. In the light of the earlier findings in Bao et al, Bao & Fukuhara chose to introduce a single duplication of KlPDIl on the host chromosome.
  • a method for producing a desired protein comprising providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene (optionally the third gene being recombinant) encoding the desired protein (optionally a heterologous protein), wherein the first and second chaperones are different; and culturing the host cell in a culture medium under conditions that allow the expression of the first, second and third genes.
  • the thus expressed desired protein may. or may not, be purified from the cultured host cell or the culture medium.
  • the thus purified desired protein may. or may not. be lyophilised.
  • the method may, or may not. further comprise the step of formulating the purified desired protein with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form, in the manner discussed above.
  • the term '"recombinant gene' includes nucleic acid sequences that operate independently as “stand alone' “ expressible sequences to produce an encoded protein or, in the alternative, nucleic acid sequences introduced that operate in combination with endogenous sequences (such as by integration into an endogenous sequence so as to produce a nucleic acid sequence that is different to the endogenous sequence) within the host to cause increased expression of a target protein.
  • a ''recombinant gene is typically a gene that is not naturally found in the context used.
  • a gene that is integrated, at an integration site, into the chromosome of a host organism can be said to be a ''recombinant gene" if it comprises a sequence that does not naturally occur at the integration site.
  • the "recombinant gene” may, or may not, comprise a non-natural sequence in the coding, regulatory or any other region of the gene, or may, or may not, comprise the sequence of a naturally occurring gene but be introduced into the chromosome of a host organism at an integration site at which that sequence does not naturally occur.
  • the same issues apply, mutatis mutandis, to the insertion of a "recombinant gene" into a plasmid.
  • chromosomaily integrated and "integrated into the chromosome of the host cell” are well recognised terms of the art. For avoidance of doubt, these terms include the integration of polynucleotide sequences in any inheritable nuclear material that naturally occurs in a host cell, other than for naturally occurring plasmids.
  • a polynucleotide sequence that is "integrated into the chromosome of the host cell” may. or may not. be integrated into the chromosome of a procaryotic (such as a bacterial) cell, or into any part of the genome of a eucaryotic cell, such as into nuclear genetic material including the chromosome (or, one of the chromosomes), the mitochondrial genome or the chloroplast genome.
  • the first and second chaperones may, or may not. each individually, be one of the specifically listed chaperones as discussed below, and are a combination of chaperones that, when co-expressed in the same host cell, provide at least an additive effect to the increase in expression of the desired protein.
  • additive effect we mean that the level of expression of the desired protein in the host cell is higher when the first and second recombinant genes are simultaneously co- expressed with the third gene as compared to the same system wherein (i) the first recombinant gene is co-expressed with the third gene in the absence of the expression of the second recombinant gene and (ii) the second recombinant gene is co-expressed with the third gene in the absence of the expression of the first recombinant gene.
  • chaperone refers to a protein that binds to and stabilises an otherwise unstable conformer of another protein, and by controlled binding and release, facilitates its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Accordingly a chaperone is also a protein that is involved in protein folding, or which has chaperone activity or is involved in the unfolded protein response.- Chaperone proteins of this type are known in the art, for example in the Stanford Genome Database (SGD), http://db.yeastgenome.org or http://www.yeastgenome.org. Preferred chaperones are eucaryotic chaperones, especially preferred chaperones are yeast chaperones, including AHAl, CCT2,
  • a chaperone useful in the practice of the present invention may. or may not, be:
  • a heat shock protein such as a protein that is a member of the hsp70 family of proteins (including Kar2p, SSA and SSB proteins, for example proteins encoded by SSAL SSA2, SSA3, SSA4, SSBl and SSB2), a protein that is a member of the HSP90-family, or a protein that is a member of the HSP40-family or proteins involved in their modulation (e.g. SiI Ip). including DNA-J and DNA-J-like proteins (e.g. Jemlp, Mdj2p);
  • a protein that is a member of the karyopherin/importin family of proteins such as the alpha or beta families of karyopherin/importin proteins, for example the karyopherin beta protein PSEl ;
  • a protein that acts in the cytosol such as the hsp70 proteins, including SSA and SSB proteins, for example protein production SSAJ, SSA2, SSA3, SSA4, SSBl and SSB2;
  • a protein that is "essential' " to the viability of the cell such as PDI
  • protein disulphide isomerases e.g. Pdilp, Mpdlp
  • homologues e.g. Euglp
  • related proteins e.g. Mpd2p, Fmolp, Erolp
  • a protein that binds preferentially or exclusively to unfolded, rather than mature protein such as the hsp70 proteins, including SSA and SSB proteins, for example proteins encoded by SSAl, SSA2, SSA3, SSA4.
  • SSA and SSB proteins for example proteins encoded by SSAl, SSA2, SSA3, SSA4.
  • a protein that prevents aggregation of precursor proteins in the cytosol such as the hsp70 proteins, including SSA and SSB proteins, for example proteins encoded by SSAl, SSA2, SSA3, SSA4, SSBl and SSB2:
  • a protein that is involved in the unfolded protein response or provides for increased resistance to agents (such as tunicamycin and dithiothreitol) that induce the unfolded protein response such as a member of the ORMDL family described by Hjelmqvist et al, 2002, supra (for example. 0rm2p) or proteins involved in the response to stress (e.g. Ubi4p);
  • a protein that is a co-chaperone and/or a protein indirectly involved in protein folding and/or the unfolded protein response e.g. hsplO4p, Mdj lp
  • a protein that is a peptidyl-prolyl cis-trans isomerases e.g. Cpr3p, Cpr ⁇ p
  • a protein that is a mitochondrial chaperone e.g Cpr3p
  • a protein that is a cytoplasmic or nuclear chaperone e.g Cnslp
  • a protein that is a membrane-bound chaperone e.g. 0rm2p, Fmolp
  • chaperone activator activity or chaperone regulatory activity e.g. Ahalp, Haclp, Hchlp
  • a protein that transiently binds to polypeptides in their immature form to cause proper folding transportation and/or secretion including proteins required for efficient translocation into the endoplasmic reticulum (e.g. Lhslp) or their site of action within the cell (e.g. Pselp);
  • a protein that is a involved in protein complex assembly and/or ribosome assembly e.g. Atpl lp, Pselp, Noblp
  • a protein of the chaperonin T-complex e.g. Cct2p
  • a protein of the prefoldin complex e.g. Pfdlp
  • a mitochondrial intermembrane space protein such as Tim9p
  • a protein that can be a functional constituent of the mitochondrial import motor associated with presequence translocase (along with Ssclp, Tim44p, Mgelp and Paml ⁇ p) such as Paml ⁇ p;
  • a preferred chaperone is protein disulphide isomerase (PDI) or a fragment or variant thereof having an equivalent ability to catalyse the formation of disulphide bonds within the lumen of the endoplasmic reticulum (ER).
  • PDF we include any protein having the ability to reactivate the ribonuclease activity against RNA of scrambled ribonuclease as described in EP 0 746 61 1 and Hillson el al, 1984, Methods En ⁇ ymol, 107, 281-292.
  • PDl is an enzyme which typically catalyzes thiokdisulphide interchange reactions, and is a major resident protein component of the ER lumen in secretory cells.
  • a body of evidence suggests that it plays a role in secretory protein biosynthesis (Freedman, 1984, Trends Biochem. Sci., 9, 438-41) and this is supported by direct cross-linking studies in situ (Roth and Pierce, 1987, Biochemistry, 26, 4179-82).
  • the finding that microsomal membranes deficient in PDI show a specific defect in cotranslational protein disulphide (Bulleid and Freedman. 1988. Nature, 335, 649- 51) implies that the enzyme functions as a catalyst of native disulphide bond formation during the biosynthesis of secretory and cell surface proteins.
  • the deletion or inactivation of the endogenous PDI gene in a host results in the production of an inviable host.
  • the endogenous PDI gene is an '"essential " ' gene.
  • PDI is readily isolated from mammalian tissues and the homogeneous enzyme is a homodimer (2x57 kD) with characteristically acidic pi (4.0-4.5) (Hillson el al, 1984, op. cit.).
  • the enzyme has also been purified from wheat and from the alga Chlamydomonas reinhardii (Kaska et al, 1990. Biochem. J, 268, 63-68), rat (Edman et al, 1985, Nature, 317, 267-270), bovine (Yamauchi et al, 1987, Biochem. Biophys. Res. Comm., 146, 1485-1492).
  • Preferred PDI sequences include those from humans and those from yeast species, such as 5. cerevisiae.
  • a yeast protein disulphide isomerase precursor, PDIl can be found as Genbank accession no. CAA42373 or BAA00723 and has a sequence of 522 amino acids as described in WO 2005/061718, the contents of which are incorporated herein by reference.
  • An alternative yeast protein disulphide isomerase sequence can be found as Genbank accession no. CAA38402, which has a sequence of 530 amino acids as described in WO 2005/061718. the contents of which are incorporated herein by reference.
  • variants and fragments of the above PDI sequences, and variants of other naturally occurring PDI sequences are also included in the present invention.
  • a "variant”, in the context of PDI refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.
  • enzymatic activity type of and specific activity
  • thermostability activity in a certain pH-range
  • conservative substitutions is intended combinations such as VaI, He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Tip.
  • Preferred conservative substitutions include GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • a “variant” typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the polypeptide from which it is derived.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
  • a '"fragment in the context of PDI, refers to a protein wherein at one or more positions there have been deletions.
  • the fragment may. or may not. comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%, more preferably up to 90%, even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature PDI protein.
  • Particularly preferred fragments of PDI protein comprise one or more whole domains of the desired protein.
  • a fragment or variant of PDl may, or may not, be a protein that, when expressed recombinant!)' in a host cell, can complement the deletion of the endogenously encoded PDI gene in the host cell, such as S. cerevisiae, and may, or may not, for example, be a naturally occurring homolog of PDI, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
  • the second chaperone is not an hsp70 chaperone protein (such as yeast KAR2, HSP70, BiP, SSA1-4, SSBl , SSCl, SSDl or a eucaryotic hsp70 protein such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP), glucose-regulated proteins 75, 78 and 80
  • hsp70 chaperone protein such as yeast KAR2, HSP70, BiP, SSA1-4, SSBl , SSCl, SSDl or a eucaryotic hsp70 protein such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP), glucose-regulated proteins 75, 78 and 80
  • the first chaperone is not yeast PDI when the second chaperone is yeast KAR2.
  • the first chaperone is not mammalian PDI when the second chaperone is mammalian BiP.
  • the first and second chaperones are, for example. PDI, particularly mammalian or yeast PDl, and an hsp70 chaperone protein as described above, respectively, then the desired protein may be a heterologous protein that may or may not be a protein selected from -
  • mammalian gene products such as enzymes, cytokines, growth factors, hormones, vaccines, antibodies and the like; erythropoietin, insulin, somatotropin, growth hormone releasing factor, platelet derived growth factor, epidermal growth factor, transforming growth factor ⁇ , transforming growth factor ⁇ , epidermal growth factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor I, insulin-like growth factor II, clotting Factor VIII, superoxide dismutase, ⁇ -interferon, ⁇ -interferon, interleukin-1, interleukin-2, interleukin-3, interleukin-4.
  • HIV human immunodeficiency virus
  • rickettsii vaccinia, Shigella, poliovirus, adenovirus, influenza, hepatitis A, hepatitis B, dengue virus, Japanese B encephalitis, Varicella zostei ⁇ cytomegalovirus, hepatitis A, rotavirus, as well as vaccines against viral diseases like Lyme disease, . measles, yellow fever, mumps, rabies, herpes, influenza, parainfluenza and the like; or bacteria such as Vibrio cholerae, Salmonella typhi, Bordetella pertussis, Streptococcus pneumoniae, Hemophilus influenza, Clostridium tetcmi. Corynebacferium diphtheriae, Mycobacterium leprae, ⁇ eisseriaqonorrhoeae, Neissei ⁇ ameningitidis, Coccidioides immitis and the like.
  • Another preferred chaperone is a protein comprising the sequence of a protein encoded by the gene SSAl, or a fragment or variant thereof having an equivalent chaperone-like activity.
  • SSAl also known as YGlOO, is located on chromosome I of the S. cerevisiae genome and is 1.93-kbp in size.
  • the protein Ssalp belongs to the Hsp70 family of proteins and is resident in the cytosol. Hsp70s possess the ability to perform a number of chaperone activities; aiding protein synthesis, assembly and folding; mediating translocation of polypeptides to various intracellular locations, and resolution of protein aggregates (Becker & Craig, 1994, Eur. J. Biochem. 219, 11-23). Hsp70 genes are highly conserved, possessing an N-terminal ATP -binding domain and a C- terminal peptide-binding domain. Hsp70 proteins interact with the peptide backbone of, mainly unfolded, proteins. The binding and release of peptides by hsp70 proteins is an ATP-dependent process and accompanied by a conformational change in the hsp70 (Becker & Craig, 1994, supra).
  • Cytosolic hsp70 proteins are particularly involved in the synthesis, folding and secretion of proteins (Becker & Craig, 1994, supra). In S. cerevisiae cytosolic hsp70 proteins have been divided into two groups; SSA (SSA 1-4) and SSB (SSB
  • SSA proteins are particularly involved in posttranslational biogenesis and maintenance of precursors for translocation into the endoplasmic reticulum and mitochondria (Kim el ai, 1998, Proc. Natl. Acad. ScI USA. 95, 12860-12865; Ngosuwan et al., 2003, J. Biol. Chem. 278 (9), 7034-7042).
  • conservative substitutions are intended combinations such as VaI. He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Trp.
  • Preferred conservative substitutions include GIy, Ala; VaI. He. Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg: and Phe, Tyr.
  • a "variant" of Ssalp typically has at least 25%. at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Ssalp.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
  • a '"fragment in the context of Ssalp, refers to a protein having the sequence of native Ssalp other than for at one or more positions where there have been deletions.
  • the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%. more preferably up to 90%, even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature Ssalp protein.
  • Particularly preferred fragments of SSAl protein comprise one or more whole domains of the desired protein.
  • a fragment or variant of Ssalp may. or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenously encoded SSAl gene (or homolog thereof) in the host cell and may, or may not. for example, be a naturally occurring homolog of Ssalp, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
  • Another preferred chaperone is protein comprising the sequence of a protein encoded by the PSEJ gene, or a fragment or variant thereof having equivalent chaperone-like activity.
  • PSEl also known as KAPl 2
  • KAPl 2 is an essential gene, located on chromosome XIII.
  • the PSEl gene is 3.25-kbp in size. Pselp is involved in the nucleocytoplasmic transport of macromolecuies (Seedorf & Silver, 1997, Proc. Natl. Acad. Sci. USA. 94, 8590-8595). This process occurs via the nuclear pore complex (NPC) embedded in the nuclear envelope and made up of nucleoporins (Ryan & Wente, 2000. Curr. Opin. Cell Biol. 12, 361-371). Proteins possess specific sequences that contain the information required for nuclear import, nuclear localisation sequence (NLS) and export, nuclear export sequence (NES) (Pemberton et al., 1998, Curr. Opin. Cell Biol. 10, 392-399).
  • NPC nuclear pore complex
  • Pselp is a karyopherin/importin, a group of proteins, which have been divided up into ⁇ and ⁇ families.
  • Karyopherins are soluble transport factors that mediate the transport of macromolecules across the nuclear membrane by recognising NLS and NES, and interact with and the NPC (Seedorf & Silver, 1997, supra; Pemberton el al, 1998, supra; Ryan & Wente, 2000, supra). Translocation through the nuclear pore is driven by GTP hydrolysis, catalysed by the small GTP -binding protein, Ran (Seedorf & Silver, 1997, supra).
  • Pselp has been identified as a karyopherin ⁇ . 14 karyopherin ⁇ proteins have been identified in S.
  • Pselp is localised to the nucleus, at the nuclear envelope, and to a certain extent to the cytoplasm. This suggests the protein moves in and out of the nucleus as part of its transport function (Seedorf & Silver, 1997, supra). Pselp is involved in the nuclear import of transcription factors (Isoyama el al.,
  • Pselp protein secretion enhancer
  • variants and fragments of Pselp are also included in the present invention.
  • a “variant' " in the context of Pselp, refers to a protein having the sequence of native Pselp other than for at one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.
  • “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
  • conservative substitutions are intended combinations such as VaI, He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Tip.
  • Preferred conservative substitutions include GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • a '"variant" of Pselp typically has at least 25%. at least 50%. at least 60% or at least 70%. preferably at least 80%. more preferably at least 90%. even more preferably at least 95%. yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Pselp.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
  • a '"fragment in the context of Pselp, refers to a protein having the sequence of native Pselp other than for at one or more positions where there have been deletions.
  • the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%, more preferably up to 90%. even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature Pselp protein.
  • Particularly preferred fragments of Pselp protein comprise one or more whole domains of the desired protein.
  • a fragment or variant of Psel p may, or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenous PSEl gene in the host cell and may, or may not, for example, be a naturally occurring homolog of Pselp, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
  • Another preferred chaperone is a protein comprising the sequence of a protein encoded by the ORA42 gene, or a fragment or variant thereof having equivalent chaperone-like activity.
  • ORM2. also known as YLR350W. is located on chromosome XII (positions 828729 to 829379) of the S. cerevisiae genome and encodes an evolutionarily conserved protein with similarity to the yeast protein Ormlp. Hjelmq ⁇ 'ist el al, 2002, Genome Biology, 3(6), research 0027.1-0027.16 reports that 0RM2 belongs to gene family comprising three human genes (ORMDLl, ORMDL2 and ORMDL3) as well as homologs in microsporidia, plants. Drosophila, urochordates and vertebrates. The ORMDL genes are reported to encode transmembrane proteins anchored in the proteins endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • Orm2p is required for resistance to agents that induce the unfolded protein response.
  • Hjelmqvist et al, 2002 reported that a double knockout of the two S. cerevisiae ORMDL homologs (ORMl and ORMI) leads to a decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol.
  • Orm2p One published sequence of Orm2p is as described in WO 2005/061718, the contents of which are incorporated herein by reference.
  • the above protein is encoded in S. cerevisiae by the coding nucleotide sequence also as described in WO 2005/061718, the contents of which are incorporated herein by reference, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical protein product.
  • variants and fragments of Orm2p are also included in the present invention.
  • 0rm2p other than for at one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.
  • enzymatic activity type of and specific activity
  • thermostability activity in a certain pH-range
  • conservative substitutions include GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn. GIn; Ser. Thr; Lys, Arg; and Phe, Tyr.
  • a "variant" of Orm2p typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Orm2p.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may, or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
  • fragment in the context of Orm2p, refers to a protein having the sequence of native Orm2p other than for at one or more positions where there have been deletions.
  • the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or
  • Orm2p protein 99% of the complete sequence of the full mature Orm2p protein.
  • Particularly preferred fragments of Orm2p protein comprise one or more whole domains of the desired protein.
  • a fragment or variant of 0rm2p may, or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenous 0RM2 gene in the host cell and may, or may not, for example, be a naturally occurring homolog of 0rm2p, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
  • a gene encoding a protein comprising the sequence of a chaperone may, or may not, be formed in a like manner to that discussed below for genes encoding heterologous proteins, with particular emphasis on combinations of ORFs and regulatory regions.
  • one preferred chaperone is protein disulphide isomerase; another preferred chaperone is 0rm2p or a fragment or variant thereof.
  • the first and second chaperones are protein disulphide isomerase and 0rm2p or a fragment or variant thereof.
  • first and second chaperones may, or may not, be encoded by the genes AHAl and CCT2; AHAl and CCT3; AHAl and CCT4 ⁇ AHAl and CCT5; AHAl and CCT6; AHAl and CCT7; AHAl and CCT8; AHAl and CNSl; AHAl and CPR3; AHAl and CPR6; AHAl and EROl: AHAl and EUGl; AHAl and FMOl; AHAl and HCHl; AHAl and HSPlO; AHAl and HSPl 2; AHAl and HSP104; AHAl and HSP26; AHAl and HSP30; AHAl and HSP42; AHAl and HSP60; AHAl and HSP78; AHAl and HSP82; AHAl and JEMl; AHAl and MDJl; AHAl and MDJ2; AHAl and MPDl; AHAl and MPD2; AHAl and MPD2; A
  • CCT2 and CAW CC77 and CPR 3; CCT2 and CP7?5; CCT2 and £7? 07; CCT2 and EUGl; CCT2 and FMOl; CCT2 and HCHl; CCT2 and HSPlO; CCT2 and HSP 12; CCT2 and HSP 104; CCT2 and HSP26; CCT2 and HSP30; CCT2 and HSP 42; CCT2 and HSP60; CCT2 and #SP7S; CCT2 and HSP82: CCT2 and J£M7; CCT2 and MZ)Ji; CCT2 and MDJ2; CCT2 and MPD7; COT and MPD2; CC72 and PDIl ; CC7L?
  • CCT6 and ORMl CCT6 and ORM2
  • CCT6 and PERl CCT6 and PTC2: CCT6 and PSEl; CCT 6 and UBI4; CCT6 and HACl or a truncated intronless PL4CT; CC77 and CCT8; CCTl and CTVSi; CCTl and CPTU; CCP7 and CPR6; CCTl and £7?O7; CCP7 and EUGl; CCTl and FM07; CCTl and PO77: CCP7 and HSPlO; CCTl and HSP72; CCTl and HSP 104; CCTl and 7PSP26; CC77 and /75PJ0; CCP7 and HSP 42; CCTl and /7SP ⁇ 50; CCTl and TP5P7S; CCP7 and H
  • HCHl and HSP704 HCHl and H5P26; HCH/ and HSP30; HCHl and 7-/5P ⁇ 2;
  • HCH/ and HSP60 HCHl and H5P7S; HCHl and H5P52; HCH/ and JEMl;
  • HCH/ and PDIl HCHl and PFDl; HCH/ and ABCl; HCHl and ⁇ PJ/; HCHl and ATPIl; HCHl and .47P/2; HCHl and 5777; HCHl and C/3C57; HCH/ and
  • H5P70 and HSP104 HSPlO and HSP26; HSPlO and HSP30; HSPlO and HSP42;
  • HSPlO and HSP60 HSPlO and H5P75; H5P70 and HSP82: HSPlO and J£M7;
  • HSPlO and CPR7 HSPlO and H5CS2; H5P70 and KAR2; HSPlO and LHSl;
  • HSPlO and SSAl HSPlO and SSAl; HSPlO and SSA2; HSPlO and 55 ⁇ 3; HSPlO and 55 ⁇ 4; 7/5P70 and 55C7; HSPlO and 55£2; H5P70 and 5/17; HSPlO and 515/; HSPlO and
  • HSP12 and HSP104 HSP12 and Z/5P26; //5P/2 and HSP30; HSPl 2 and //5P ⁇ 2;
  • HSP 12 and PFD/ //5P72 and PDIl; HSP 12 and PFD/ ; HSP 12 and ⁇ /3C7; //5P/2 and .4PJ/ ; HSP12 and ATP11; HSP12 and ATP12; HSP12 and BTT1: HSP12 and CDC37; HSP 12 and CPR7; HSP 12 and HSC82; HSP 12 and KAR2: HSP 12 and LHS1; HSP 12 and MGE1; HSP 12 and MRS11; HSP 12 and NOB1; HSP 12 and ECM10; HSP 12 and SSA1; HSP12 and SSA2; HSP 12 and SSA3; HSP 12 and SSA4; HSP12 and SSC1; HSP12 and SSE2; HSP 12 and SIL1: HSP12 and SLS1; HSP 12 and ORM1; HSP 12 and ORM2; HSP 12 and PER1; HSP12 and PTC2; HSP 12 and PSE1; HSP12 and UBI
  • HSP26 and H5C52; 775P25 and TC4T?2 HSP26 and Z//57; HSP26 and MG£7; 7/5P25 and M7?577; HSP26 and NO737; HSP 26 and £CM70; 7J5P2d and SSAl; HSP26 and SS42; H5P25 and 55 ⁇ 43; HSP26 and SS ⁇ ; HSP26 and 55C7; 775P25 and 55£2; HSP 26 and 5717; 775P25 and 5L57; HSP26 and O7?i/7; HSP 26 and OT?M2; H5P2(5 and P£7?7; HSP26 and P7C2; 7/5P25 and P5£7; 775P25 and UBU; HSP26 and 7£4Ci or a truncated intronless HACl; HSP30 and HSP42; J ⁇ SP30 and /75P50; HSP 30 anfr HSP78; HSP30 and MG
  • HSP30 and MDJl T75P30 and MDJl; HSP30 and MDJ2; T75P30 and AfPDi; HSP30 and AfPD2; HSP30 and PDIl; HSP 30 and PFDJ: HSP 30 and ABCl; HSP30 and APJl: HSP30 and ATPIh.
  • HSP78 and ⁇ ZP77 //5P7S and ⁇ 7P72; HSP78 and 7377/; HSP78 and CDC37: HSP78 and CPR7; HSP 78 and HSC82: HSP78 and KAR2; HSP 78 and LHSl: HSP 78 and MGEh HSP 78 and MRSIl; HSP78 and NOBl: HSP78 and £CAfi0; //5P 75 and SSAl: HSP78 and S&42; HSP 78 and 5S ⁇ 3; /75P7S and SSA4; HSP78 and 55Ci; 7/5P7, ⁇ and SSE2; HSP78 and 5717; HSP78 and 5L57; /75P7S and ORMl: HSP78 and ORM2; HSP78 and / > £/?/; 77SP75 and PTC2; HSP78 and P5£7; 7/5P7S and UBI4; HSP78 and
  • AfDJi and ORMl MDJl and 0RM2; MDJl and P£7?7; AfDJ/ and PTC2; MDJl and PSEl: MDJl and UB14; MDJl and HACl or a truncated intronless HACl: MDJ2 and MPDl; MDJ2 and MPD2: MDJ2 and PDIJ; MDJ2 and PFDl: MDJ2 and ,4P>C7; MDJ2 and _4PJ7; M ⁇ /2 and ATPIl: MDJ2 and .477V 2; MDJ2 and BTTl; MDJ2 and CDC37; MDJ2 and CPi?
  • MDJ2 and HSC82 MDJ2 and /C4T?2; MDJ2 and Z//5T ; MDJ2 and MG£7 ; MDJ2 and MRSl 1 ; MDJ2 and NOBl : MDJ2 and ECMlO: MDJ2 and SSAl: MDJ2 and SSA2: MDJ2 and 5&43; MDJ2 and S&44; MDJ2 and SSCl; MDJ2 and 55£2; M73J2 and SILI; MDJ2 and 5LST; MDJ2 and 0T?MT; MDJ2 and 0RM2; MDJ2 and P£T?7; MDJ2 and PTC2: MDJ2 and P5£T; MDJ2 and f/T3/4; MDJ2 and HACl or a truncated intronless HACl; MPDT and MPD2; MPDl and PD/T ; MPDl and PP 1 DT ; MPDT and ABCl ; MPDT and APJl
  • SSAl ATP12 and 55 ⁇ 2; ⁇ 7P72 and SSA3; ATP12 and 55.44; ⁇ PP72 and 55C7; ATP] 2 and SSE2: ATPl 2 and SILJ: ATP 12 and SLSl: ATP12 and ORMl-, ATP12 and ORM2; ATP12 and PERl: ATP12 and PTC2: ATP12 and PSEl; ATP12 and £7574; -4.TP72 and HACl or a truncated intronless HACl; BTTl and CDCJ7; BTTl and CPi?
  • BTTl and 7C4T?2 5777 and LHSl; BTTl and MGEl; BTTl and M?577; 73777 and NOBl; BTTl and ECMlO; BTTl and 5547; 5777 and SSA2; BTTl and S5L43; 73777 and SSA4: BTTl and 55C7; 73777 and SSE2; BTTl and SILl; BTTl and 5L57: 73777 and ORMl; BTTl and ORM2; 5777 and PERl; BTTl and P7C2; 5777 and PSEl; BTTl and UBM; BTTl and 7I4C7 or a truncated intronless 7i4C7; CDC37 and CPT?
  • TCPl and MGZ/ 7CPi and MRSIl; TCPl and .VOJ?/; 7CPi and ECMlO; TCPl and SSAl; TCPl and SS42; 7CPi and SSA3; TCPl and SS ⁇ 4; 7CPi and SSCl; TCPl and SSE2; TCP!
  • the first, second and third recombinant genes may, or may not. each individually be present on a plasmid within the host cell (which may, or may not, be a 2 ⁇ m- family plasmid, as discussed above) or be chromosomally integrated within the genome of the host cell.
  • first, second and third recombinant genes may be used.
  • the first, second and third recombinant genes may, or may not, each individually be present on a plasmid, and this may, or may not, be either the same plasmid or different plasmids.
  • the first recombinant gene may, or may not, be present on a plasmid
  • second and third recombinant genes may, or may not, be chromosomally integrated within the genome of the host cell.
  • the first and second recombinant genes may.
  • the third recombinant gene may, or may not, be chromosomally integrated within the genome of the host cell.
  • the first and third recombinant genes may, or may not, be present on a plasmid and the second recombinant gene may, or may not, be chromosomally integrated within the genome of the host cell.
  • the first and second recombinant gene may, or may not. be chromosomally integrated within the genome of the host cell and the third recombinant gene may, or may not, be present on a plasmid.
  • the first, second and third recombinant genes may, or may not, each individually be chromosomally integrated within the genome of the host cell.
  • Plasmids used for this purpose may, or may not, be plasmids, such as 2 ⁇ m-family plasmids, as defined below.
  • a method according to the first aspect of the invention does not involve a host cell in which the first, second and third recombinant genes are all present on the 2 ⁇ m-family plasmid.
  • the present invention also provides a plasmid wherein the plasmid comprises two different genes (the first and second recombinant genes) encoding different chaperones.
  • the plasmid may. or may not. further comprise a gene encoding a heterologous protein (the third recombinant gene), such as a heterologous protein as described above.
  • a plasmid according to the second aspect of the invention may, or may not, be a 2 ⁇ m-family plasmid.
  • a third aspect of the present invention provides for the use of the plasmid of the second aspect of the invention as an expression vector to increase the production of a desired protein, including as heterologous protein, such as a fungal (optionally yeast) or vertebrate protein.
  • the desired protein may, or may not, be encoded by a recombinant gene that is present as part of the plasmid, or present in the host cell on a different plasmid, or present in the host cell as a transgene that is integrated in the host cell's chromosome.
  • a fourth aspect of the invention provides a host cell comprising a plasmid as defined above.
  • the host cell may, or may not, further comprise a recombinant gene encoding a desired heterologous protein.
  • the recombinant gene that encodes the desired heterologous protein (the "third recombinant gene") is not present as part of the same plasmid that encodes the first and second chaperones, then the host cell may, or may, not, comprise the third recombinant gene on a different plasmid, or as a transgene that is integrated in the host cell's chromosome.
  • the present invention provides a host cell which comprises the first, second and third recombinant genes.
  • the first, second and third recombinant genes may, or may not, each individually be present on a plasmid within the host cell (which may, or may not, be a 2 ⁇ m-family plasmid, as discussed above) or be chromosomally integrated within the genome of the host cell. It will be appreciated that any combination of plasmid and chromosomally integrated first, second and third recombinant genes may be used . , as discussed above.
  • the host cell may, or may not, comprise the first, second and third recombinant genes each individually present on a plasmid, and this may, or may not.
  • the host cell may. or may not, comprise the first recombinant gene on a plasmid, and second and third recombinant genes chromosomally integrated within the genome of the host cell.
  • the host cell may, or may not, comprise the first and second recombinant genes on a plasmid and the third recombinant gene chromosomally integrated within the genome of the host cell.
  • the host cell may. or may not, comprise the first and third recombinant genes on a plasmid and the second recombinant gene chromosomally integrated within the genome of the host cell.
  • the host cell may, or may not, comprise the first and second recombinant genes chromosomally integrated within the genome of the host cell and the third recombinant gene present on a plasmid.
  • the host cell may, or may not, comprise the first, second and third recombinant genes each individually chromosomally integrated within the genome of the host cell.
  • a plasmid may, or may not, be a 2 ⁇ m- family plasmid.
  • Certain closely related species of budding yeast have been shown to contain naturally occurring circular double stranded DNA plasmids.
  • These plasmids collectively termed 2 ⁇ m-family plasmids, include pSRl, pSB3 and pSB4 from Zygosaccharomyces roiaii (formerly classified as Zygosaccharomyces bisporus), plasmids pSBl and pSB2 from Zygosaccharomyces bailii, plasmid pSMl from Zygosaccharomyces ferment ati, plasmid pKDl from Kluyveromyces drosphilarum, an un-named plasmid from Pichia membranaefaciens (hereinafter "pPMl”) and the 2 ⁇ m plasmid (such as shown in Figure
  • 2 ⁇ m-family plasmids may, or may not, be used in the present invention, but this invention is not limited to the use of naturally occurring 2 ⁇ m- family plasmids.
  • a 2 ⁇ m-family plasmid may, or may not, be as described below.
  • a 2 ⁇ m-family plasmid is a circular, double stranded, DNA plasmid. It is typically small, such as between 3,000 to 10,000 bp. optionally between 4,500 to 7000 bp, excluding recombinantly inserted sequences.
  • a 2 ⁇ m-family plasmid typically comprises at least three open reading frames (“ORFs") that each encodes a protein that functions in the stable maintenance of the 2 ⁇ m-family plasmid as a multicopy plasmid.
  • ORFs open reading frames
  • the proteins encoded by the three ORFs can be designated FLP, REPl and REP2.
  • ORFs encoding the missing protein(s) should be supplied in trans, either on another plasmid or by chromosomal integration.
  • a "FLP" protein is a protein capable of catalysing the site-specific recombination between inverted repeat sequences recognised by FLP.
  • the inverted repeat sequences are termed FLP recombination target (FRT) sites and each is typically present as part of a larger inverted repeat (see below).
  • Preferred FLP proteins comprise the sequence of the FLP proteins encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl , pPMl and the 2 ⁇ m plasmid, for example as described in Volkert et al, op cit., Murray et al, op. cit., and Painting el al.
  • FLP proteins are also included in the present invention.
  • "Fragments” and '"variants” are those which retain the ability of the native protein to catalyse the site-specific recombination between the same FRT sequences.
  • Such variants and fragments will usually have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with an FLP protein encoded by one of plasmids pSRl, pSBl, pSB2 ; pSB3, pSB4, pSML pKDl, pPMl and the 2 ⁇ m plasmid.
  • Different FLP proteins can have different FRT sequence specificities.
  • a typical FRT site may, or may not, comprise a core nucleotide sequence flanked by inverted repeat sequences.
  • the FRT core sequence is 8 nucleotides in length and the flanking inverted repeat sequences are 13 nucleotides in length (Volkert et al, op. cit.).
  • the FRT site recognised by any given FLP protein may, or may not, be different to the 2 ⁇ m plasmid FRT site.
  • REPl and REP2 are proteins involved in the partitioning of plasmid copies during cell division, and may, or may not, also have a role in the regulation of FLP expression. Considerable sequence divergence has been observed between REPl proteins from different 2 ⁇ m-family plasmids, whereas no sequence alignment is possible between REP2 proteins derived from different 2 ⁇ m-family plasmids.
  • Preferred REPl and REP 2 proteins comprise the sequence of the REPl and REP 2 proteins encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2 ⁇ m plasmid, for example as described in Volkert et al, op. cit. , Murray et al, op. cit. , and Painting et al, op. cit. Variants and fragments of these REPl and REP2 proteins are also included in the present invention.
  • “Fragments” and “variants” of REPl and REP2 are those which, when encoded by the plasmid in place of the native ORP. do not substantially disrupt the stable multicopy maintenance of the plasmid within a suitable yeast population. Such variants and fragments of REPl and REP 2 will usually have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with a REPl and REP2 protein, respectively, as encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2 ⁇ m plasmid.
  • REPl and REP2 proteins encoded by the ORFs on the plasmid must be compatible. It is preferred that the REPl and REP2 proteins have the sequences of REPl and REP2 proteins encoded by the same naturally occurring 2 ⁇ m-family plasmid, such as pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2 ⁇ m plasmid, or variant or fragments thereof.
  • a 2 ⁇ m-family plasmid typically comprises two inverted repeat sequences.
  • the inverted repeats may be any size, so long as they each contain an FRT site (see above).
  • the inverted repeats are typically highly homologous. They may, or may not, share greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,
  • sequence identity In a preferred embodiment they are identical.
  • the inverted repeats are each between 200 to 1000 bp in length.
  • Preferred inverted repeat sequences may, or may not, each have a length of from
  • inverted repeats are those of the plasmids pSRl (959 bp), pSBl (675 bp), pSB2 (477 bp), pSB3
  • sequences of the inverted repeats may, or may not, be varied.
  • sequences of the FRT site in each inverted repeat should be compatible with the specificity of the FLP protein encoded by the plasmid, thereby to enable the encoded FLP protein to act to catalyse the site-specific recombination between the inverted repeat sequences of the plasmid. Recombination between inverted repeat sequences (and thus the ability of the FLP protein to recognise the FRT sites with the plasmid) can be determined by methods known in the art.
  • a plasmid in a yeast cell under conditions that favour FLP expression can be assayed for changes in the restriction profile of the plasmid which would result from a change in the orientation of a region of the plasmid relative to another region of the plasmid.
  • the detection of changes in restriction profile indicate that the FLP protein is able to recognise the FRT sites in the plasmid and therefore that the FRT site in each inverted repeat are compatible with the specificity of the FLP protein encoded by the plasmid.
  • sequences of inverted repeats, including the FRT sites are derived from the same 2 ⁇ m-family plasmid as the ORF encoding the FLP protein, such as pSRl, pSBl. pSB2, pSB3, pSB4, pSMl , pKDl. pPMl or the 2 ⁇ m plasmid.
  • the inverted repeats are typically positioned with the 2 ⁇ m-family plasmid such that the two regions defined between the inverted repeats (e.g. such as defined as UL and US in the 2 ⁇ m plasmid) are of approximately similar size, excluding exogenously introduced sequences such as transgenes.
  • one of the two regions may, or may not, have a length equivalent to at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, up to 100%, of the length of the other region.
  • a 2 ⁇ m-family plasmid typically comprises the ORF that encodes FLP and one inverted repeat (arbitrarily termed ''IRl" to distinguish it from the other inverted repeat mentioned in the next paragraph) juxtaposed in such a manner that IRl occurs at the distal end of the FLP ORF, without any intervening coding sequence, for example as seen in the 2 ⁇ m plasmid.
  • ''IRl inverted repeat
  • IRl inverted repeat
  • a 2 ⁇ m-family plasmid typically comprises the ORF that encodes REP2 and the other inverted repeat (arbitrarily termed "IR2" to distinguish it from IRl mentioned in the previous paragraph) juxtaposed in such a manner that IR2 occurs at the distal end of the REP 2 ORF, without any intervening coding sequence, for example as seen in the 2 ⁇ m plasmid.
  • IR2 inverted repeat
  • the ORFs encoding REP2 and FLP may, or may not, be present on the same region of the two regions defined between the inverted repeats of the 2 ⁇ m-family plasmid, which region may be the bigger or smaller of the regions (if there is any inequality in size between the two regions).
  • the ORFs encoding REP2 and FLP may, or may not, be transcribed from divergent promoters.
  • the regions defined between the inverted repeats (e.g. such as defined as UL and US in the 2 ⁇ m plasmid) of a 2 ⁇ m-family plasmid may. or may not, comprise not more than two endogenous genes that encode a protein that functions in the stable maintenance of the 2 ⁇ m-family plasmid as a multicopy plasmid.
  • one region of the plasmid defined between the inverted repeats may, or may not, comprise not more than the ORFs encoding FLP and REP 2; FLP and REPl; or REPl and REP 2, as endogenous coding sequence.
  • a 2 ⁇ m-family plasmid typically comprises an origin of replication (also known as an ''autonomously replicating sequence - "ARS"), which is typically bidirectional. Any appropriate ARS sequence can be present. Consensus sequences typical of yeast chromosomal origins of replication may, or may not. be appropriate (Broach et ⁇ l, 1982 ; Cold Spring Harbor Symp. Quant. Biol, 47, 1165-1174; Williamson, Yeast, 1985, 1, 1-14). Preferred ARSs include those isolated from pSRL pSBl, pSB2, pSB3 ; pSB4, pSML pKDl, pPMl and the 2 ⁇ m plasmid.
  • a preferred 2 ⁇ m-family plasmid may. or may not. comprise ORFs encoding FLP, REPl and REP2, two inverted repeat sequences each inverted repeat comprising an FRT site compatible with the encoded FLP protein, and an ARS sequence.
  • the FRT sites are derived from the same 2 ⁇ m-family plasmid as the sequence of the encoded FLP protein. More preferably the sequences of the encoded REPl and REP2 proteins are derived from the same 2 ⁇ m-family plasmid as each other. Even more preferably, the FRT sites are derived from the same 2 ⁇ m-family plasmid as the sequence of the encoded FLP, REPl and REP2 proteins.
  • sequences of the ORFs encoding FLP, REPl and REP2, and the sequence of the inverted repeats (including the FRT sites) are derived from the same 2 ⁇ m-family plasmid.
  • the ARS site may, or may not, be derived from the same 2 ⁇ m-family plasmid as one or more of the ORFs of FLP, REPl and REP 2, and the sequence of the inverted repeats (including the FRT sites).
  • an FLP gene having a sequence derived from the FLP gene of the 2 ⁇ m plasmid may, or may not, have a modified promoter or other regulator ⁇ ' sequence compared to that of the naturally occurring gene.
  • an FLP gene having a sequence derived from the FLP gene of the 2 ⁇ m plasmid may, or may not, have a modified nucleotide sequence in the open reading frame which may, or may not, encode the same protein as the naturally occurring gene, or may, or may not, encode a modified FLP protein.
  • the same considerations apply to other sequences on a 2 ⁇ m-family plasmid having a sequence derived from a particular source.
  • a 2 ⁇ m-family plasmid may, or may not, comprise a region derived from the STB region (also known as REP3) of the 2 ⁇ m plasmid, as defined in Volkert et al, op. cit.
  • the STB region in a 2 ⁇ m -family plasmid of the invention may, or may not, comprise two or more tandem repeat sequences, such as three, four, five or more. Alternatively, no tandem repeat sequences may be present.
  • the tandem repeats may be any size, such as 10, 20, 30, 40, 50, 60 70, 80, 90, 100 bp or more in length.
  • the tandem repeats in the STB region of the 2 ⁇ m plasmid are 62 bp in length.
  • sequences of the tandem repeats are not essential for the sequences of the tandem repeats to be identical. Slight sequence variation can be tolerated. It may, or may not, be preferable to select an STB region from the same plasmid as either or both of the REPl and REP2 ORFs.
  • the STB region is thought to be a cw-acting element and preferably is not transcribed.
  • a 2 ⁇ m-family plasmid may, or may not, comprise an additional ORF that encodes a protein that functions in the stable maintenance of the 2 ⁇ m-family plasmid as a multicopy plasmid.
  • the additional protein can be designated RAF or D.
  • ORFs encoding the RAF or D gene can be seen on, for example, the 2 ⁇ m plasmid and pSMl.
  • a RAF or D ORF can comprise a sequence suitable to encode the protein product of the RAF or D gene ORFs encoded by the 2 ⁇ m plasmid or pSMl, or variants and fragments thereof.
  • variants and fragments of the protein products of the RAF or D genes of the 2 ⁇ m plasmid or pSMl are also included in the present invention.
  • “Fragments” and “variants” of the protein products of the RAF or D genes of the 2 ⁇ m plasmid or pSMl are those which, when encoded by the 2 ⁇ m plasmid or pSMl in place of the native ORF, do not disrupt the stable multicopy maintenance of the plasmid within a suitable yeast population.
  • Such variants and fragments will usually have at least 5%, 10%, 20%, 30%, 40%, 50%, 60% ; 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with the protein product of the RAF or D gene ORFs encoded by the 2 ⁇ m plasmid or pSMl .
  • a naturally occurring 2 ⁇ m-family plasmid may, or may not. be preferred.
  • a naturally occurring 2 ⁇ m-family plasmid is any plasmid having the features defined above, which plasmid is found to naturally exist in yeast, i.e. has not been recombinantly modified to include heterologous sequence.
  • the naturally occurring 2 ⁇ m-family plasmid is selected from pSRl (Accession No. X02398), pSB3 (Accession No. X02608) or pSB4 as obtained from Zygosaccharomyces rouxii, pSBl or pSB2 (Accession No.
  • NC_002055 or Ml 8274 both as obtained from Zygosaccharomyces bailli
  • pSMl Accession No. NC_002054
  • pKDl obtained from Zygosaccharomyces fermentati
  • pPMl from Pichia membranaefaciens
  • 2 ⁇ m plasmid accession numbers in this paragraph refer to NCBI deposits.
  • the 2 ⁇ m plasmid ( Figure 1) is a 6,318-bp double-stranded DNA plasmid, endogenous in most Saccharomyces cerevisiae strains at 60-100 copies per haploid genome.
  • the 2 ⁇ m plasmid comprises a small unique (US) region and a large unique (UL) region, separated by two 599-bp inverted repeat sequences. Site-specific recombination of the inverted repeat sequences results in inter- conversion between the A-form and B-form of the plasmid in vivo (Volkert & Broach, 1986, Cell, 46, 541).
  • the two forms of 2 ⁇ m differ only in the relative orientation of their unique regions.
  • Saccharomyces cerevisiae gave a size of 6,318-bp (Hartley and Donelson, 1980,
  • ⁇ 'ariation gene polymorphism occurs within the natural population of 2 ⁇ m plasmids found in S. cerevisiae and S. carlsbergensis, with the NCBI sequence (accession number NC_001398) being one example.
  • the 2 ⁇ m plasmid has a nuclear localisation and displays a high level of mitotic stability (Mead et al, 1986, Molecular & General Genetics, 205, 417).
  • the inherent stability of the 2 ⁇ m plasmid results from a plasmid-encoded copy number amplification and partitioning mechanism, which can be compromised during the development of chimeric vectors (Futcher & Cox. 1984, J. Bacleriol, 157, 283; Bachmair & Ruis, 1984, Monatshefte fur Chemie, 115, 1229).
  • a yeast strain, which contains a 2 ⁇ m plasmid is known as [cir + ]
  • a yeast strain which does not contain a 2 ⁇ m plasmid is known as [cir 0 ].
  • the US-region of the 2 ⁇ m plasmid contains the REP2 and FLP genes, and the UL-region contains the REPl and D (also known as RAF) genes, the STB-locus and the origin of replication (Broach & Hicks, 1980, Cell, 21, 501; Sutton &
  • FIp Recognition Target within the inverted repeats to mediate site-specific recombination, which is essential for natural plasmid amplification and control of plasmid copy number in vivo (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A.,
  • Repl and Rep2 proteins mediate plasmid segregation, although their mode of action is unclear (Sengupta et al, 2001, J. Bacteriol, 183, 2306). They also repress transcription of the FLP gene (Reynolds et al, 1987, Mo/. Cell Biol, 7, 3566).
  • the FLP and REP2 genes of the 2 ⁇ m plasmid are transcribed from divergent promoters, with apparently no intervening sequence defined between them.
  • the FLP and REP2 transcripts both terminate at the same sequence motifs within the inverted repeat sequences, at 24-bp and 178-bp respectively after their translation termination codons (Sutton & Broach, 1985, MoJ. Cell. Biol, S, 2770).
  • the C-terminal coding sequence also lies within the inverted repeat sequence.
  • the two inverted repeat sequences are highly conserved over 599-bp, a feature considered advantageous to efficient plasmid replication and amplification in vivo, although only the FRT-sites (less than 65- bp) are essential for site-specific recombination in vitro (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 7270; Jayaram, 1985, Proc. Natl. Acad. Sci.
  • Residues 15-58 form a Repl- binding domain, and residues 59-296 contain a self-association and STB-binding region (Sengupta et al, 2001, J. Bacteriol, 183, 2306).
  • Chimeric or large deletion mutant derivatives of 2 ⁇ m which lack many of the essential functional regions of the 2 ⁇ m plasmid but retain the functional cis element ARS and STB, cannot effectively partition between mother and daughter cells at cell division.
  • Such plasmids can do so if these functions are supplied in trans, by for instance the provision of a functional 2 ⁇ m plasmid within the host, such as a [cir + ] host.
  • plasmid pSACSUl in EP 0 286 424 and the plasmid shown in Figure 2 of WO 2005/061718, which includes a ⁇ -lactamase gene (for ampicillin resistance), a LEU2 selectable marker and an oligonucleotide linker, the latter two of which are inserted into a unique SnaBl- ⁇ Xt within the UL-region of the 2 ⁇ m-like disintegration vector, pSAC3 (see EP 0 286 424).
  • pSAC3 see EP 0 286 424.
  • plasmids pCV19, pCV20, CV neo which utilise an insertion at FcoRI in FZP
  • plasmids pCV21, pGT41 and pYE which utilise EcoRl in D as the insertion site
  • plasmid pHKB52 which utilises Pstl in D as the insertion site
  • plasmid pJDB248 which utilises an insertion at Pstl in D and EcoRl in D
  • plasmid p.TDB219 in which Pstl in D and EcoRl in FLP are used as insertion sites
  • plasmid Gl 8 8, plasmid pAB18 which utilises an insertion at CIcA in FiP
  • plasmids pGT39 and pA3 plasmids p
  • 2 ⁇ m plasmids include pSAC3, ⁇ SAC3Ul, pSAC3U2, pSAC300, pSAC310, pSAC3Cl, pSAC3PLl, pSAC3SL4, and pSAC3SCl are described in EP 0 286 424 and Chinery & Hinchliffe (1989, Curr. Genet, 16, 21-25) which also described Pstl, Eagl or SnctBl as appropriate 2 ⁇ m insertion sites. Further 2 ⁇ m plasmids include pAYE255.
  • pAYE316, pAYE443, pAYE522 (Kerry-Williams et al, 1998, Yeast, 14, 161 -169), pDB2244 (WO 00/44772), and pAYE329 (Sleep et al, 2001, Yeast, 18, 403-421).
  • one or more genes are inserted into a 2 ⁇ m-family plasmid within an untranscribed region around the ARS sequence.
  • the untranscribed region around the ARS sequence extends from the end of the D gene to the beginning of ARS sequence. Insertion into SnaBI (near the origin of replication sequence ARS) is described in Chinery & Hinchliffe, 1989, Curr. Genet., 16, 21-25. The skilled person will appreciate that gene insertions can also be made in the untranscribed region at neighbouring positions to the SnaBI site described in Chinery & Hinchliffe.
  • REP2 and FLP genes in a 2 ⁇ m-family plasmid each have an inverted repeat adjacent to them, and one or more genes are inserted into a 2 ⁇ m-family plasmid within the region between the first base after the last functional codon of either the REP2 gene or the FLP gene and the last base before the FRT site in the inverted repeat adjacent to said gene.
  • the last functional codon of either a REP2 gene or a FLP gene is the codon in the open reading frame of the gene that is furthest downstream from the promoter of the gene whose replacement by a stop codon will lead to an unacceptable loss of multicopy stability of the plasmid, as defined herein.
  • the REP2 gene of the 2 ⁇ m plasmid can be disrupted after codon 59 and that the FLP gene of the 2 ⁇ m plasmid can be disrupted after codon 344, each without a loss of multicopy stability of the plasmid.
  • the last functional codon in equivalent genes in other 2 ⁇ m-family piasmids can be determined routinely by making mutants of the piasmids in either the FLP or REP 2 genes and following the tests set out herein to determine whether the plasmid retains multicopy stability.
  • a plasmid insertion site as defined in WO 2005/061719 may, or may not, be used to carry one or more a recombinant genes according to any aspect of the present invention.
  • Plasmid stability may be defined as the percentage cells remaining prototrophic for the selectable marker after a defined number of generations. The number of generations will preferably be sufficient to show a difference between a control plasmid. such as pSAC35 or pSACSIO, or to shown comparable stability to such a control plasmid.
  • the number of generations may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more. Higher numbers are preferred.
  • the acceptable plasmid stability might be 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or substantially 100%. Higher percentages are preferred.
  • the skilled person will appreciate that, even though a plasmid may have a stability less than 100% when grown on non-selective media, that plasmid can still be of use when cultured in selective media.
  • plasmid pDB2711 as described in the examples is only 10% stable when the stability is determined accordingly to test of Example 2 of WO 2005/061719, but provides a 15-fold increase in recombinant transferrin productivity in shake flask culture under selective growth conditions.
  • one or more gene insertions may. or may not, occur between the first base after the last functional codon of the REP2 gene and the last base before the FRT site in an inverted repeat adjacent to said gene, preferably between the first base of the inverted repeat and the last base before the FRT site, more preferably at a position after the translation termination codon of the REP2 gene and before the last base before the FRT site.
  • one or more gene insertions may, or may not. occur between the first base after the last functional codon of the FLP gene and the last base before the FRT site in an inverted repeat adjacent to said gene, preferably between the first base of the inverted repeat and the last base before the FRT site, more preferably between the first base after the end of the FLP coding sequence and the last base before the FRT site, such as at the first base after the end of the FLP coding sequence.
  • the 2 ⁇ m-family plasmid is based on the 2 ⁇ m plasmid of S cerevisiae, it is a disintegration vector as known in the art (for example, see EP 286 424. the contents of which are incorporated herein by reference).
  • a disintegration vector may, or may not, be a 2 ⁇ m plasmid vector comprising a DNA sequence which is intended to be lost by recombination, three
  • sequence to be lost may, or may not, comprise a selectable marker DNA sequence.
  • a preferred disintegration vector may, or may not, comprise a complete 2 ⁇ m plasmid additionally carrying (i) a bacterial plasmid DNA sequence necessary for propagation of the ⁇ 'ector in a bacterial host; (ii) an extra 2 ⁇ m FRT site; and a selectable marker DNA sequence for yeast transformation; the said bacterial plasmid DNA sequence being present and the extra FRT site being created at a restriction site, such as Xbal, in one of the two inverted repeat sequences of the 2 ⁇ m plasmid.
  • a particularly preferred 2 ⁇ m plasmid vector has substantially the configuration of pSAC3 as shown in EP 286 424.
  • disintegration vector as used herein also includes plasmids as defined in US 6,451,559. the contents of which are incorporated herein by reference.
  • a disintegration vector may, or may not, be a 2 ⁇ m vector that, other than DNA sequence encoding non-yeast polypeptides, contains no bacterial (particularly E. coif) origin of replication, or more preferably no bacterial (particularly E. coli) sequence and preferably all DNA in said vector, other than DNA sequence encoding non-yeast polypeptides, is yeast-derived DNA.
  • Desired proteins and other proteins defined by the present application are desirable proteins and other proteins defined by the present application:
  • protein and “desired protein” as used herein includes all natural and non-natural proteins, polypeptides and peptides.
  • a “heterologous protein” is a protein that is encoded by a “recombinant gene” as described above.
  • the “heterologous protein” may, or may not, be identical in sequence to a protein that is encoded by one of more other genes that naturally occur in the expression system that is used (by “expression system” we include the meaning of a host cell's genome (typically the chromosome) where the “recombinant gene” is chromosomally integrated, or a plasmid where the "recombinant gene” is encoded by a plasmid).
  • heterologous protein that is encoded by a “recombinant gene” carried on a 2 ⁇ m- family plasmid
  • the "heterologous protein'” may. or may not. be a protein that is not naturally encoded by a 2 ⁇ m-family plasmid and can also be described as a "non 2 ⁇ m-family plasmid protein”.
  • heterologous protein and “non 2 ⁇ m-family plasmid protein” are used synonymously in this application.
  • the heterologous protein when encoded by a 2 ⁇ m-family, is not a FLP, REPl, REP 2, or a RAF/D protein as encoded by any one of pSRl, pSB3 or pSB4 as obtained from Z. rouxii, pSBl or pSB2 both as obtained from Z. bailli, pSMl as obtained from Z fermentati, pKDl as obtained from K. drosophilarum, pPMl as obtained from P. membranaefaciens or the 2 ⁇ m plasmid as obtained from S. cerevisiae.
  • a gene encoding a desired heterologous, or other, protein comprises a polynucleotide sequence encoding the heterologous protein (typically according to standard codon usage for any given organism), designated the open reading frame ("ORF").
  • the gene may, or may not, additionally comprise some polynucleotide sequence that does not encode an open reading frame (termed “non-coding region").
  • Non-coding region in the gene may, or may not, contain one or more regulatory sequences, operatively linked to the ORF, which allow for the transcription of the open reading frame and/or translation of the resultant transcript.
  • regulatory sequence refers to a sequence that modulates (i.e., promotes or reduces) the expression (i.e., the transcription and/or translation) of an ORF to which it is operably linked.
  • Regulatory regions typically include promoters, terminators, ribosome binding sites and the like. The skilled person will appreciate that the choice of regulatory region will depend upon the intended expression system. For example, promoters may, or may not, be constitutive or inducible and may, or may not. be cell- or tissue -type specific or non-specific. Suitable regulatory regions, may, or may not. be 5bp. lObp, 15bp. 20bp, 25bp.
  • the gene encoding a chaperone may, or may not, additionally comprise non-coding regions and/or regulatory regions.
  • non-coding regions and regulatory regions are not restricted to the native non-coding regions and/or regulatory regions normally associated with the chaperone ORP.
  • suitable promoters for S. cerevisiae include those associated with the PGKl gene, GALl or GALlO genes, TEFl 1 TEF2, PYKl, PMAl, CYCl, PHO5, TRPl, ADHl, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase.
  • ⁇ -mating factor pheromone a-mating factor pheromone
  • the PRBl promoter the PRAl promoter
  • the GPDl promoter the promoter of the PRBl promoter
  • hybrid promoters involving hybrids of parts of 5' regulator ⁇ ' regions with parts of 5' regulatory regions of other promoters or with upstream activation sites e.g. the promoter of EP-A-258 067.
  • Suitable transcription termination signals are well known in the art. Where the host cell is eucaryotic, the transcription termination signal is optionally derived from the 3' flanking sequence of a eucaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, or may not, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may, or may not, correspond to the promoter. Alternatively, they may be different. In that case, and where the host is a yeast, optionally S. cerevisiae. then the termination signal of the S. cerevisiae ADHl, ADH2, CYCL, or PGKl genes are preferred.
  • the promoter and open reading frame of the gene such as a gene encoding the chaperone (e.g. PDIl) or a desired protein (such as a heterologous desired protein), to be flanked by transcription termination sequences so that the transcription termination sequences are located both upstream and downstream of the promoter and open reading frame, in order to prevent transcriptional read-through into neighbouring genes, such as 2 ⁇ m genes, and vice versa.
  • a gene encoding the chaperone e.g. PDIl
  • a desired protein such as a heterologous desired protein
  • the favoured regulatory sequences in yeast include: a yeast promoter (e.g. the Saccharomyces cerevisiae PRBl promoter), as taught in EP 431 880; and a transcription terminator, optionally the terminator from Saccharomyces ADHl, as taught in EP 60 057.
  • the vector incorporates at least two translation stop codons.
  • non-coding region may incorporate more than one DNA sequence encoding a translational stop codon, such as UAA, UAG or UGA, in order to minimise translational read-through and thus avoid the production of elongated, non-natural fusion proteins.
  • a translational stop codon such as UAA, UAG or UGA
  • operably linked includes within its meaning that a regulatory sequence is positioned within any non-coding region in a gene such that it forms a relationship with an ORF that permits the regulatory region to exert an effect on the ORP in its intended manner.
  • ORF is positioned in such a way that the regulator ⁇ ' region is able to influence transcription and/or translation of the ORF in the intended manner, under conditions compatible with the regulatory sequence.
  • the desired protein (such as the heterologous desired protein) is secreted.
  • a sequence encoding a secretion leader sequence which, for example, comprises most of the natural HSA secretion leader, plus a small portion of the S cerevisiae ⁇ -mating factor secretion leader as taught in WO 90/01063 may, or may not, be included in the open reading frame.
  • the desired protein (such as a heterologous desired protein) may, or may not, be intracellular.
  • the desired protein may, or may not, comprise the sequence of a eucaryotic protein, or a fragment or variant thereof.
  • Suitable eucaryotes include fungi, plants and animals.
  • the heterologous protein may, or may not, be a fungal protein, such as a yeast protein.
  • the desired protein (such as a heterologous desired protein) may, or may not, be an animal protein.
  • Exemplary animals include vertebrates and invertebrates.
  • Exemplar ⁇ ' vertebrates include mammals, such as humans, and non-human mammals.
  • the desired protein may, or may not, comprise the sequence of a yeast protein. It may. or may not, for example, comprise the sequence of a yeast protein from the same host from which a 2 ⁇ m- family plasmid is derived, particularly if the gene encoding the heterologous protein is integrated into said 2 ⁇ m-family plasmid.
  • a method, use or plasmid of the invention may, or may not, comprise DNA sequences encoding more than one heterologous protein, more than one chaperone, or more than one heterologous protein and more than one chaperone.
  • the desired protein may, or may not, comprise the sequence of albumin, a monoclonal antibody, an etoposide. a serum protein (such as a blood clotting factor), antistasin, a tick anticoagulant peptide, transferrin, lactoferrin. endostatin. angiostatin. collagens, immunoglobulins or immunoglobulin-based molecules or fragment of either (e.g.
  • 'SMIP Small Modular ImmunoPharmaceuticalTM
  • dAb Fab' fragments, F(ab')2, scAb, scFv or scFv fragment
  • Kunitz domain protein such as those described in WO 03/066824, with or without albumin fusions
  • interferons interleukins, ILlO, ILI l, IL2, interferon ⁇ species and sub- species, interferon ⁇ species and sub-species, interferon ⁇ species and sub-species, leptin, CNTF, CNTF Ax is, ILl -receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5- helix, T20 peptide, Tl 249 peptide, HIV gp41, HIV gpl20, urokinas
  • EPO
  • prothrombin von Willebrand's factor, o. ⁇ -antitrypsin, plasminogen activators, Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, or a variant or fragment of any of the above.
  • PD-ECGF platelet-derived endothelial cell growth factor
  • glucose oxidase glucose oxidase
  • serum cholinesterase serum cholinesterase
  • aprotinin amyloid precursor protein
  • inter-alpha trypsin inhibitor antithrombin III
  • apo-lipoprotein species Protein C
  • Protein S Protein S
  • a “variant”, in the context of the above-listed proteins, refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity or receptor binding (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed.
  • “Significantly” in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
  • conservative substitutions are intended combinations such as VaI, lie. Leu. Ala. Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe. Tyr, Trp.
  • Preferred conservative substitutions include GIy, Ala; VaI, He, Leu; Asp. GIu: Asn. GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • a '-variant typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the polypeptide from which it is derived.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res., 22(22), 4673-80).
  • the parameters used may, or may not, be as follows:
  • fragment in the context of the above-listed proteins, refers to a protein wherein at one or more positions there have been deletions. Thus the fragment may, or may not. comprise at most 5. 10. 20, 30, 40 or 50% of the complete sequence of the full mature polypeptide. Typically a fragment comprises up to 60%, more typically up to
  • Particularly preferred fragments of a protein comprise one or more whole domains of the protein.
  • the desired protein (such as a desired heterologous protein) comprises the sequence of albumin or a variant or fragment thereof.
  • albumin we include a protein comprising the sequence of an albumin protein obtained from any source. Typically the source is mammalian.
  • the serum albumin is human serum albumin ("HSA").
  • HSA human serum albumin
  • the term “human serum albumin'” includes the meaning of a serum albumin having an amino acid sequence naturally occurring in humans, and variants thereof.
  • the albumin has the amino acid sequence disclosed in WO 90/13653 or a variant thereof.
  • the HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 73 646 and EP 286 424.
  • the "albumin” comprises the sequence of bovine serum albumin.
  • bovine serum albumin includes the meaning of a serum albumin having an amino acid sequence naturally occurring in cows, for example as taken from Swissprot accession number P02769, and variants thereof as defined below.
  • bovine serum albumin' also includes the meaning of fragments of full-length bovine serum albumin or variants thereof, as defined below.
  • the albumin comprises the sequence of an albumin derived from one of serum albumin from dog (e.g. see Swissprot accession number P49822), pig (e.g. see Swissprot accession number P08835), goat (e.g. as available from Sigma as product no. A2514 or A4164), turkey (e.g. see Swissprot accession number 073860), baboon (e.g. as available from Sigma as product no. Al 516), cat (e.g. see Swissprot accession number P49064), chicken (e.g. see Swissprot accession number P19121), ovalbumin (e.g. chicken ovalbumin) (e.g. see Swissprot accession number POl 012), donkey (e.g.
  • Swissprot accession number P39090 guinea pig (e.g. as available from Sigma as product no. A3060, A2639, 05483 or A6539), hamster (e.g. as available from Sigma as product no. A5409), horse (e.g. see Swissprot accession number P35747), rhesus monkey (e.g. see Swissprot accession number Q28522), mouse (e.g. see Swissprot accession number O89020).
  • pigeon e.g. as defined by Khan et al, 2002, Int. J. Biol. Macromol, 30(3-4),171-8
  • rabbit e.g. see Swissprot accession number P49065
  • rat e.g. see Swissprot accession number P36953
  • sheep e.g. see Swissprot accession number P14639 and includes variants and fragments thereof as defined below.
  • albumin Many naturally occurring mutant forms of albumin are known. Many are described in Peters. (1996, All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, Inc., San Diego, California, p.170-181). A variant as defined above may, or may not, be one of these naturally occurring mutants.
  • a “variant albumin” refers to an albumin protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolality (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH- stability) has not significantly been changed. "'Significantly " in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
  • an albumin variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with naturally occurring albumin.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson el ah, 1994). The parameters used may, or may not, be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; I 5 window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.
  • fragment includes any fragment of full-length albumin or a variant thereof, so long as at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed.
  • binding activity type of and specific activity e.g. binding to bilirubin
  • osmolarity oncotic pressure, colloid osmotic pressure
  • behaviour in a certain pH-range pH-range
  • a fragment wall typically be at least 50 amino acids long.
  • a fragment may, or may not, comprise at least one whole sub-domain of albumin.
  • HSA HSA proteins also comprises six sub-domains (sub-domains IA, IB, HA, HB, IHA and IHB).
  • Sub-domain IA comprises amino acids 6-105
  • sub-domain IB comprises amino acids 120-177
  • sub-domain HA comprises amino acids 200-291
  • sub-domain HB comprises amino acids 316-369
  • sub-domain IIIA comprises amino acids 392-491
  • sub-domain HIB comprises amino acids 512-583.
  • a fragment may, or may not, comprise a whole or part of one or more domains or sub-domains as defined above, or any combination of those domains and/or sub-domains.
  • the desired protein (such as a desired heterologous protein) comprises the sequence of transferrin or a variant or fragment thereof.
  • transferrin includes all members of the transferrin family (Testa, Proteins of iron metabolism, CRC Press, 2002; Harris & Aisen, Iron carriers and iron proteins, Vol. 5, Physical Bioinorganic Chemistry, VCH, 1991) and their derivatives, such as transferrin, mutant transferrins (Mason et al, 1993, Biochemistry, 32, 5472; Mason et al, 1998, Biochem. J, 330(1), 35), truncated transferrins, transferrin lobes (Mason et al, 1996, Protein Expr. PuHf.
  • the transferrin may, or may not, be human transferrin.
  • human transferrin is used herein to denote material which is indistinguishable from transferrin derived from a human or which is a variant or fragment thereof.
  • a "variant” includes insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the useful ligand- binding or immunogenic properties of transferrin.
  • Mutants of transferrin are included in the invention. Such mutants may, or may not, have altered immunogenicity.
  • transferrin mutants may, or may not, display modified (e.g. reduced) glycosylation.
  • the N-linked glycosylation pattern of a transferrin molecule can be modified by adding/removing amino acid glycosylation consensus sequences such as N-X-S/T, at any or all of the N, X, or S/T position.
  • Transferrin mutants may, or may not, be altered in their natural binding to metal ions and/or other proteins, such as transferrin receptor.
  • An example of a transferrin mutant modified in this manner is exemplified below.
  • variants or fragments of human transferrin will have at least 5%, 10%, 15%, 20%, 30%, 40% or 50% (preferably at least 80%, 90% or 95%) of human transferrin's ligand binding activity (for example iron-binding), weight for weight.
  • the iron binding activity of transferrin or a test sample can be determined spectrophotometrically by 470nm:280nm absorbance ratios for the proteins in their iron-free and fully iron-loaded states. Reagents should be iron-free unless stated otherwise.
  • Iron can be removed from transferrin or the test sample by dialysis against 0.1M citrate, 0.1 M acetate, 1OmM EDTA pH4.5. Protein should be at approximately 20mg/mL in 10OmM HEPES, 1OmM NaHCO 3 pH8.0. Measure the 470nm:280nm absorbance ratio of apo- transferrin (Calbiochem, CN Biosciences, Nottingham, UK) diluted in water so that absorbance at 280nm can be accurately determined spectrophotometrically (0% iron binding).
  • single or multiple heterologous fusions comprising any of the above; or single or multiple heterologous fusions to albumin, transferrin or immunoglobins or a variant or fragment of any of these may, or may not.
  • Such fusions include albumin N-terminal fusions, albumin C-terminal fusions and co-N-terminal and C-terminal albumin fusions as exemplified by WO 01/79271, and transferrin N-terminal fusions, transferrin C-terminal fusions, and co-N- terminal and C-terminal transferrin fusions.
  • transferrin fusions are given in US patent applications US2003/0221201 and US2003/0226155, Shin, et al., 1995, Proc Natl Acad Sci USA, 92, 2820, AIi, et al, 1999, J Biol Chem, 214, 24066, Mason, et al., 2002, Biochemistry, 41, 9448. the contents of which are incorporated herein by reference.
  • the open reading frame of any other gene or variant, or part or either can be utilised as an open reading frame for use with the present invention.
  • the open reading frame may, or may not, encode a protein comprising any sequence, be it a natural protein (including a zymogen), or a variant, or a fragment (which may, or may not, for example, be a domain) of a natural protein; or a totally synthetic protein; or a single or multiple fusion of different proteins (natural or synthetic).
  • Such proteins can be taken, but not exclusively, from the lists provided in WO 01/79258, WO 01/79271, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, or a variant or fragment thereof; the disclosures of which are incorporated herein by reference.
  • these patent applications present the list of proteins in the context of fusion partners for albumin, the present invention is not so limited and, for the purposes of the present invention, any of the proteins listed therein may, or may not, be presented alone or as fusion partners for albumin, the Fc region of immunoglobulin, transferrin, lactoferrin or an)' other protein or fragment or variant of any of the above, as a desired polypeptide.
  • the desired protein (such as a desired heterologous protein) may, or may not, be a therapeutically active protein. In other words, it may, or may not, have a recognised medical effect on individuals, such as humans. Many different types of therapeutically active protein are well known in the art.
  • the desired protein (such as a desired heterologous protein) may, or may not, be a protein that is useful in diagnostic techniques. Many different types of diagnostically useful protein are well known in the art.
  • the desired protein (such as a desired heterologous protein) may, or may not, be a protein that has no relationship to healthcare. It may, or may not, for example, be a protein that has a utility as an industrial, domestic or nutritional (e.g. as a foodstuff or additive) agent. Many different types of proteins having industrial, domestic and/or nutritional utilities are also well known in the art.
  • the desired protein (such as a desired heterologous protein) may, or may not, comprise a leader sequence effective to cause secretion in a host cell, such as in a yeast cell.
  • the signal sequence directs the nascent protein towards the machinery of the cell that exports proteins from the cell into the surrounding medium or. in some cases, into the periplasmic space.
  • the signal sequence is usually, although not necessarily, located at the N-terminus of the primary translation product and is generally, although not necessarily, cleaved off the protein during the secretion process, to yield the '"mature" protein.
  • the entity that is initially secreted, after the removal of the signal sequence includes additional amino acids at its N-terminus called a "pro" sequence, the intermediate entity being called a "pro-protein".
  • pro sequences may, or may not, assist the final protein to fold and become functional, and are usually then cleaved off.
  • the pro region simply provides a cleavage site for an enzyme to cleave off the pre-pro region and is not known to have another function.
  • the pro sequence can be removed either during the secretion of the protein from the cell or after export from the cell into the surrounding medium or periplasmic space.
  • leader sequences Polypeptide sequences which direct the secretion of proteins, whether they resemble signal (i.e. pre) sequences or pre-pro secretion sequences, are referred to as leader sequences.
  • the secretion of proteins is a dynamic process involving translation, translocation and post-translational processing, and one or more of these steps may not necessarily be completed before another is either initiated or completed.
  • leader sequences include those from the S. cerevisiae acid phosphatase protein (Pho5p) (see EP 366 400), the invertase protein (Suc2p) (see Smith et al.
  • Hspl50p heat-shock protein-150
  • MFa-1 S. cerevisiae mating factor alpha- 1 protein
  • HSA human serum albumin
  • WO 90/01063 discloses a fusion of the MFa- 1 and HSA leader sequences, which advantageously reduces the production of a contaminating fragment of human albumin relative to the use of the MFa- 1 leader sequence.
  • leader sequences are also disclosed in WO 2004/009819 and in the examples of this application; the reader will appreciate that those leader sequences can be used with proteins other than transferrin.
  • the natural transferrin leader sequence may, or may not, be used to direct secretion of transferrin and other heterologous proteins.
  • a chaperone that is recombinantly expressed according to the present invention is protein disulphide isomerase
  • the desired protein may, or may not, comprise disulphide bonds in its mature form. Any disulphide bonds may, or may not, be intramolecular and/or intermolecular.
  • the desired protein may. or may not, be a commercially useful protein, such as a therapeutically, diagnostically, industrially, domestically or nutritionally useful protein.
  • Some proteins, such as heterologously expressed proteins are intended to interact with the cell in which they are expressed in order to bring about a beneficial effect on the cell's activities. These proteins are not, in their own right, commercially useful.
  • Commercially useful proteins are proteins that have a utility ex vivo of the cell in which they are expressed. Nevertheless, the skilled reader will appreciate that a commercially useful protein may, or may not, also have a biological effect on the host cell expressing it (such as a heterologous protein), but that that effect is not the main or sole reason for expressing the protein therein.
  • Commercially useful proteins may include proteins that are useful as metabolites or antibiotics, and the like.
  • the desired protein (such as a desired heterologous protein) is not ⁇ -lactamase. In another embodiment it is preferred that the desired protein (such as a desired heterologous protein) is not antistasin.
  • provisos exclude genes encoding either ⁇ -lactamase or antistasin from being present in a host cell or on a plasmid of the invention, merely that the gene encoding the desired protein (such as a desired heterologous protein) encodes a protein other than ⁇ -lactamase and/or antistasin.
  • Plasmids useful in the practice of the present invention can. unless specified otherwise, be any type of plasmid.
  • references to '"plasmids' " may, or may not, also include a reference to other types of vectors. It may be appropriate to choose a suitable plasmid based on the host cell system in which it will be used.
  • bacteria e.g. Bacillus subtilis or
  • Escherichia coli transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeasts (e.g. Saccharomyces cerevisiae or Pichia pastoris) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (e.g. baculovirus); plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems, either in cell culture, transgenic or as gene therapy, transfected with, for example, adenovirus expression vectors.
  • yeasts e.g. Saccharomyces cerevisiae or Pichia pastoris
  • insect cell systems transformed with, for example, viral expression vectors (e.g. baculovirus)
  • plant cell systems transfected with, for example viral or bacterial expression vectors e.g. baculovirus
  • animal cell systems either in cell culture, transgenic or as gene therapy, transfected with, for example, a
  • Typical procaryotic vector plasmids are: pUC18, pUC19 ; pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA): p7>c99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors. pNH8A, pNH16A, pNHI SA. pNH46A available from Stratagene Cloning Systems (La JoIIa, CA 92037, USA).
  • a typical mammalian cell vector plasmid is pS ⁇ L available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells.
  • An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors include the 2 ⁇ m-family plasmids (as described above), as well as pRS403-406 and pRS413-416 which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).
  • Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPJ, LEU2 and ORAS.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (YCps). Other Yips and YCps plasmids may also be used.
  • Plasmids for use in any aspect of the present invention can be prepared by modifying plasmids, such as 2 ⁇ m-family plasmids, known in the art by inserting the required sequences (for example, one or more genes encoding chaperones and/or one or more genes encoding a heterologous protein) using techniques well known in the art such as are described in by Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2001, 3rd edition, the contents of which are incorporated herein by reference.
  • one such method involves ligation via cohesive ends.
  • Compatible cohesive ends can be generated on a DNA fragment for insertion and plasmid by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
  • a further method uses synthetic double stranded oligonucleotide linkers and adaptors.
  • DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3 ' ends.
  • Synthetic linkers and pieces of blunt-ended double-stranded DNA which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini.
  • Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end. Alternath'ely a DNA fragment or DNA fragments can be ligated together by the action of DNA ligase in the presence or absence of one or more synthetic double stranded oligonucleotides optionally containing cohesive ends.
  • Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Sigma-Genosys Ltd, London Road, Pampisford, Cambridge, United Kingdom.
  • Appropriate insertion sites in plasmids include, but are not limited to, those discussed above.
  • the present invention also provides a host cell comprising recombinant genes and/or plasmid according to any aspect of the present invention.
  • the host cell may be any type of cell.
  • Many suitable host cell expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis), filamentous fungi (for example Aspergillus), plant cells, whole plants, animal cells and insect cells.
  • Bacterial and yeast host cells may, or may not, be preferred.
  • Bacterial host cells may be useful for cloning purposes.
  • Yeast host cells may be useful for expression of genes present in the plasmid.
  • the host cell may. or may not. be a yeast cell, such as a member of the Saccharomyces, Kluyveromyces, Arxula, YarroM'ia, Candida, Schi ⁇ osaccharomyces, Debaryomyces, Xanthophyllomyces, Geothchum, Ashbya, Hortaea, Schwanniomyces, T ⁇ chosporon, Xanthophyllomyces, or Pichia genus.
  • yeast cell such as a member of the Saccharomyces, Kluyveromyces, Arxula, YarroM'ia, Candida, Schi ⁇ osaccharomyces, Debaryomyces, Xanthophyllomyces, Geothchum, Ashbya, Hortaea, Schwanniomyces, T ⁇ chosporon, Xanthophyllomyces, or Pichia genus.
  • Yeast such Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Pichia membranaefaciens, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha (also known as Pichia augusta), Arxula adeninivorans, Yarrowia lipolytica, Candida boidinii Candida utilis, Schizosaccharomyces pombe may, or may not. be preferred.
  • yeast may, or may not, include Debaryomyces hansenii, Xanthophyllomyces dendrorhous, Geotrichum candidum, Ashbya gossypii, Hortaea wasneckii, SchM'anniomyces occidentalis, Trichosporon domesticum, and/or Xanthophyllomyces dendrorhous,
  • yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins for instance by disruption of the gene coding sequence, as discussed in WO 2004/083245, the contents of which are incorporated herein by reference.
  • the host cell may, or may not, be an animal cell.
  • the animal cell may, or may not, be a mammalian cell, such as a human cell type.
  • the host cell type may. or may not, be selected for compatibility with a plasmid type being used. Plasmids obtained from one yeast type can be maintained in other yeast types (Me el al, 1991 , Gene, 108(1), 139-144; lrie el al, 1991 , MoI. Gen. Genet , 225(2). 257-265). For example. pSRl from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae.
  • the host cell is compatible with a 2 ⁇ m-family plasmid (see above for a full description of the following plasmids).
  • a suitable yeast cell is Zygosaccharomyces rouxii; where the plasmid is based on pSBl or pSB2 then a suitable yeast cell is Zygosaccharomyces bailli; where the plasmid is based on pSMl then a suitable yeast cell is Zygosaccharomyces fermentati; where the plasmid is based on pKDl then a suitable yeast cell is Kluyveromyces drosophilarum; where the plasmid is based on pPMl then a suitable yeast cell is Pichia membranaefaciens; where the plasmid is based on the 2 ⁇ m plasmid then a suitable yeast cell is Pichia membranaefaciens; where the plasmid is based on the 2 ⁇ m
  • a 2 ⁇ m-family plasmid of the invention can be said to be "based on" a naturally occurring plasmid if it comprises one, two or preferably three of the genes FLP, REPl and REP 2 having sequences derived from that naturally occurring plasmid.
  • yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins may, or may not, be particularly advantageous to use a yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins, for instance by disruption of the gene coding sequence.
  • Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell.
  • the albumin protein sequence does not contain any sites for N-linked glycosylation and has not been reported to be modified, in nature, by 0-linked glycosylation.
  • rHA recombinant human albumin
  • the mannosylated albumin is able to bind to the lectin Concanavalin A.
  • the amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687).
  • the most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced.
  • a defect in its genome such that a reduced level of one of the Pmt proteins is produced.
  • the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
  • disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia past oris or Kluyveromyces lactis.
  • the sequence of PMTl (or any other PKdT gene) isolated from S. cerevisiae may, or may not, be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species.
  • the yeast may, or may not, have a deletion of the HSP 150 and/or YAPS genes as taught respectively in WO 95/33833 and WO 95/23857.
  • a plasmid as defined above may, or may not, be introduced into a host through standard techniques.
  • transformation of procaryotic host cells see, for example, Cohen et al (1972) Proc. Natl. Acad ScL USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 rd Ed. Cold Spring Harbor
  • Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cell, bacterial cells and vertebrate cells. ' Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
  • a plasmid may, or may not, comprise a selectable marker, including but not limited to bacterial selectable marker and/or a yeast selectable marker.
  • a typical bacterial selectable marker is the ⁇ -lactamase gene although many others are known in the art.
  • Yeast selectable marker include LEU2, TRPl, HIS3, HIS4, URA3, URA5, SFAl, ADE2, METIS, LYS5, LYS2, ILV2, FBAl, PSEl, PDIl and PGKl.
  • any gene whose chromosomal deletion or inactivation results in an inviable host can be used as a selective marker if a functional gene is provided on the plasmid. as demonstrated for PGKl in a pgkl yeast strain (Piper and Curran, 1990, Curr. Genet. 17. 1 19). Suitable "essential" genes can be found within the Stanford Genome Database (SGD), http:://db. yeastgenome.org). Any "essential" gene product (e.g.
  • auxotrophic (biosynthetic) requirement we include a deficiency which can be complemented by nutrient and other additions or modifications to the growth medium.
  • essential marker genes in the context of the present invention are those that, when deleted or inactivated in a host cell, result in a deficiency which cannot be complemented by any additions or modifications to the growth medium, expect where those additions or modifications are, for example, a polynucleotide, that can restore the ability of the host cell to express the product of the "essential” marker gene, or the product of the "essential” marker gene itself.
  • a plasmid as provided by, for use in a method of, or comprised in a host cell of, the present invention may, or may not, comprise more than one selectable marker.
  • One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a selectable trait in the transformed cell.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eucaryotic cell culture, and tetracyclin, kanamycin or ampicillin (i.e. ⁇ -lactamase) resistance genes for culturing in E.coli and other bacteria.
  • the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Another method of identifying successfully transformed cells involves growing the cells resulting from the introduction of the plasmid, optionally to allow the expression of a recombinant polypeptide (i.e. where a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is not naturally produced by the host).
  • a recombinant polypeptide i.e. where a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is not naturally produced by the host.
  • Cells can be harvested and lysed and their DNA or RNA content examined for the presence of the recombinant sequence using a method such as that described by Southern (1975) J. MoJ. Biol. 98, 503 or Berent el al (1985) Biotech. 3, 208 or other methods of DNA and RNA analysis common in the art.
  • the presence of a polypeptide in the supernatant of a culture of a transformed cell can be detected using antibodies.
  • transformed host cells also contemplates a culture of those cells, optionally a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • transformed cells may, or may not, represent an industrially/commercially or pharmaceutically useful product and can be used without further purification or can be purified from a culture medium and optionally formulated with a carrier or diluent in a manner appropriate to their intended industrial/commercial or pharmaceutical use, and optionally packaged and presented in a manner suitable for that use.
  • whole cells could be immobilised; or used to spray a cell culture directly on to/into a process, crop or other desired target.
  • whole cell such as yeast cells
  • whole cell such as yeast cells
  • capsules for a huge variety of applications, such as fragrances, flavours and pharmaceuticals.
  • Transfo ⁇ ned host cells may, or may not, be cultured for a sufficient time and under appropriate conditions known to those skilled in the art, and in view of the teachings disclosed herein, to permit the expression of one or more recombinant chaperones and a desired protein (such as a desired heterologous protein).
  • the culture medium may, or may not. be non-selective or may. or may not place a selective pressure on the maintenance of the plasmid.
  • the thus produced desired protein (such as a desired heterologous protein) may. or may not, be present intracellular! ⁇ ' or. if secreted, in the culture medium and/or periplasmic space of the host cell.
  • the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium optionally comprises cell immobilization, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilization, separation and/or breakage.
  • Cell immobilization techniques such as encasing the cells using calcium alginate bead, are well known in the art.
  • cell separation techniques such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like are well known in the art.
  • methods of cell breakage including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
  • the at least one other purification step may be any other step suitable for protein purification known in the art.
  • purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, US 5 728 553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference.
  • Proteins other than albumin may be purified from the culture medium by any technique that has been found to be useful for purifying such proteins.
  • Suitable methods include ammonium sulphate or ethanol precipitation, acid or solvent extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography (“HPLC”), reverse phase HPLC, conductivity adjustment and the like.
  • HPLC high performance liquid chromatography
  • any one or more of the above mentioned techniques may, or may not, be used to further purify the thus isolated protein to a commercially or industrially acceptable level of purity.
  • commercially or industrially acceptable level of purity we include the provision of the protein at a concentration of at least 0.01 g.L “1 , 0.02 g.L “1 , 0.03 g.L '1 , 0.04 g.L “1 , 0.05 g.L “ ',0.06 g.L “ ',0.07 g.1/ 1 , 0.08 g.L “ ⁇ 0.09 g.L “1 , 0.1 g.L “1 , 0.2 g.L “1 , 0.3 g.L “1 , 0.4 g.L “1 , 0.5 g.L “1 , 0.6 g.L “1 , 0.7 g.L “1 , 0.8 g.L “1 , 0.9 g.1/ 1 , 1 g.L “1 , 2
  • the desired protein (such as a desired heterologous protein) is purified to achieve a pharmaceutically acceptable level of purity.
  • a protein has a pharmaceutically acceptable level of purity if it is essentially pyrogen free and can be administered in a pharmaceutically efficacious amount without causing medical effects not associated with the activity of the protein.
  • the resulting desired protein (such as a desired heterologous protein) may, or may not, be used for any of its known utilities, which, in the case of albumin, include i.v. administration to patients to treat severe bums, shock and blood loss, supplementing culture media, and as an excipient in formulations of other proteins.
  • a therapeutically, diagnostically, industrially. domestically or nutritionally useful desired protein obtained by a process of the of the invention to be presented or administered alone
  • a formulation such as a pharmaceutical formulation, particularly in the case of therapeutically and/or diagnostically useful proteins
  • the carrier(s) or diluent(s) must be '"acceptable" in the sense of being compatible with the desired protein and, where the formulation is intended for administration to a recipient, then not deleterious to the recipient thereof.
  • the carriers or diluents will be water or saline which will be sterile and pyrogen free.
  • the thus formulated protein will be presented in a unit dosage form, such as in the form of a tablet, capsule, injectable solution or the like.
  • a method for producing a desired protein comprising: providing a host cell comprising a first recombinant gene encoding the protein comprising the sequence of 0rm2p or a variant thereof and a second gene, optionally a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), optionally with the proviso that the first and second genes are not both present within the host cell on the same 2 ⁇ m-family plasmid; and culturing the host cell in a culture medium under conditions that allow the expression of the first and second genes.
  • the method may, or may not, further comprise the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium; and optionally, lyophilising the thus purified protein; and optionally formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent; and optionally presenting the thus formulated protein in a unit dosage form.
  • desired protein such as a desired heterologous protein
  • the host cell may, or may not, further comprise a further recombinant gene encoding a protein comprising the sequence of an alternative chaperone to ⁇ rm2p or a variant thereof.
  • Either or both of the first and second genes in the sixth aspect of the invention may or may not be recombinant genes that are expressed from a plasmid, and optionally from the same plasmid, provided that where both genes are expressed from the same plasmid then that plasmid is not a 2 ⁇ m-family plasmid.
  • a further recombinant gene encoding a protein comprising the sequence of an alternative chaperone to 0rm2p or a variant thereof may, or may not, also be expressed from a plasmid, optionally from the same plasmid as either or both of the first and second recombinant genes.
  • both of the first and second genes are recombinant genes that are co-expressed from the same plasmid then either one may, or may not, be individually expressed from a 2 ⁇ m-family plasmid, such as the 2 ⁇ m plasmid.
  • one or both of the first and second genes of the sixth aspect of the invention may, or may not, be integrated into the chromosome of the host cell.
  • the further recombinant gene encoding a protein comprising the sequence of an alternative chaperone to 0rm2p or a variant thereof may, or may not, be integrated into the chromosome of the host cell, irrespective of whether or not the first and second genes are expressed from a plasmid or are chromosomally integrated.
  • the present invention also provides, in a seventh aspect, a host cell as defined above in respect of the sixth aspect, which host cell comprises a first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof and a second gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein), optionally with the proviso that the first and second genes are not present within the host cell on the same 2 ⁇ m-family plasmid.
  • a host cell as defined above in respect of the sixth aspect, which host cell comprises a first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof and a second gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein), optionally with the proviso that the first and second genes are not present within the host cell on the same 2 ⁇ m-family plasmid.
  • the present invention also provides, in an eighth aspect, for the use of a nucleic acid sequence encoding the protein 0rm2p or a variant thereof to increase the production, in a host cell (such as a host cell as defined above), of a desired protein (such as a desired heterologous protein) encoded by a gene, such as a recombinant gene, in the host cell by co-expression of the nucleic acid sequence and the gene within the host cell (but optionally not including co-expression of these genes from the same 2 ⁇ m-family plasmid). Either or both of the nucleic acid sequence and the gene encoding the desired protein may, or may not, be expressed from a plasmid within the host cell, and optionally from the same plasmid.
  • a host cell such as a host cell as defined above
  • a desired protein such as a desired heterologous protein
  • a gene such as a recombinant gene
  • the host cell may, or may not, further comprise a recombinant gene encoding an alternative chaperone to 0rm2p or a variant thereof, which may, or may not, be located on a plasmid within the host cell, optionally on the same plasmid as either or both of the nucleic acid sequence and a gene encoding the desired protein.
  • Suitable plasmids include a 2 ⁇ m-family plasmid, such as the 2 ⁇ m plasmid, as discussed above.
  • a plasmid as an expression vector to increase the production of a heterologous protein by providing a recombinant gene encoding the heterologous protein and a gene encoding Orni2p or a variant thereof on the same plasmid, optionally with the proviso that the plasmid is not a 2 ⁇ m-family plasmid.
  • the plasmid may, or may not, further comprise a gene encoding an alternative chaperone to 0rm2p or a variant thereof in the manner discussed above.
  • the present invention also provides a plasmid, optionally an expression plasmid, comprising a first gene encoding the protein Orm2p or a variant or fragment thereof and a second gene encoding a heterologous protein, as discussed above, optionally with the pro ⁇ 'iso that the plasmid is not a 2 ⁇ m-family plasmid.
  • the plasmid may. or may not, further comprise a third gene encoding an alternative chaperone to 0rm2p or a variant thereof.
  • the third gene encodes a protein comprising the sequence of protein disulphide isomerase.
  • a plasmid-borne gene encoding a protein comprising the sequence of an "essential” protein can be used to stably maintain the plasmid in a host cell that, in the absence of the plasmid. does not produce the "essential" protein.
  • This has the advantage of ensuring the genetic stability of the organism in the chosen culture conditions, and thereby improving the reproducibility and reliability of individual cultures.- and furthermore enables prolonged culture without reduced productivity due to plasmid loss.
  • a preferred "essential" protein is a chaperone which may or may not provide the further advantage that, as well as acting as a selectable marker to increase plasmid stability, its expression simultaneously increases the expression of one or more desired proteins, such as a heterologous protein encoded by a recombinant gene, within the host cell.
  • This system is advantageous because it allows the user to minimise the number of recombinant genes that need to be carried by a plasmid.
  • typical prior art plasmids carry marker genes (such as those as described above) that enable the plasmid to be stably maintained during host cell culturing process. Such marker genes need to be retained on the plasmid in addition to any further genes that are required to achieve a desired effect.
  • plasmids to incorporate exogenous DNA sequences is -limited and it is therefore advantageous to minimise the number of sequence insertions required to achieve a desired effect.
  • some marker genes (such as auxotrophic marker genes) require the culturing process to be conducted under specific conditions in order to obtain the effect of the marker gene. Such specific conditions may not be optimal for cell growth or protein production, or may require inefficient or unduly expensive growth systems to be used.
  • a recombinant gene that encodes a protein comprising the sequence of an "essential" protein as a plasmid-borne gene to increase plasmid stability, where the plasmid is present within a cell that, in the absence of the plasmid. is unable to produce the "essential" protein.
  • a protein that is not “essential” in one host organism might become “essential” when one or more other genes is deleted, disrupted, inactivated, modified or affected in that same host, and thereby be used as an "essential" plasmid-borne gene, as described above; likewise whether or not a protein is “essential” may depend on certain physical conditions, such as pH, temperature and/or oxygen levels under which the host cell is cultured.
  • the "essential protein” is one that, when its encoding gene(s) in a host cell are deleted or inactivated, does not result in the host cell developing an auxotrophic (biosynthetic) requirement.
  • auxotrophic (biosynthetic) requirement we include a deficiency that can be complemented by additions or modifications to the growth medium, in particular additions of, or modifications to, the nutrient composition of the growth medium.
  • the "essential protein” would be an auxotrophic marker protein if the inactivation of its encoding gene, in a host cell, resulted in the production of an auxotrophic mutant, i.e.
  • an "essential marker gene” which encodes an "essential protein”, in the context of the present invention is one that, when deleted or inactivated in a host cell, results in a deficiency which cannot be complemented by additions or modifications, typically nutrient additions or modifications, to the growth medium, expect where those additions or modifications are, for example, a polynucleotide, that can restore the ability of the host cell to express the product of the "essential " marker gene, or the product of the "essential” marker gene itself.
  • the ''essential protein is not a protein that, in nature, is involved in the metabolic conversion of nutrients by a host cell.
  • the advantage of this system is that the '"essential marker gene" can be used as a selectable marker on a plasmid in host cell that, in the absence of the plasmid. is unable to produce that gene product, to achieve increased plasmid stability without the disadvantage of requiring the cell to be cultured under specific selective (e.g. selective nutrient) conditions. Therefore, the host cell can be cultured under conditions that do not have to be adapted for any particular marker gene, without losing plasmid stability.
  • host cells produced using this system can be cultured in non-selective media such as complex or rich media, and under non-selective growth conditions (e.g. such as pH, temperature and/or oxygen levels), which may be more economical, and/or more supportive growth media/conditions, than the minimal media and/or specifically adapted growth conditions that are commonly used to give auxotrophic, and other, marker genes their effect.
  • non-selective media such as complex or rich media
  • non-selective growth conditions e.g. such as pH, temperature and/or oxygen levels
  • the cell may, or may not, for example, have the endogenous copy (or copies) of the gene (or genes) encoding the "essential" protein deleted or otherwise inactivated.
  • the "essential protein” is an “essential” chaperone, as this can provide the dual advantage of improving plasmid stability without the need for selective growth conditions and increasing the production of desired proteins, such as endogenously encoded or a heterologous proteins, in the host cell.
  • This system also has the advantage that it minimises the number of recombinant genes that need to be carried by the plasmid if one chooses to use over-expression of an "essential" chaperone to increase protein production by the host cell.
  • essential proteins for use in this aspect of the invention include the '"essential" chaperones encoded by the genes PDIl and PSEl which, when the endogenous gene(s) encoding these proteins are deleted or inactivated in a host cell, do not result in the host cell developing an auxotrophic (biosynthetic) requirement.
  • essential chaperones are eucaryotic chaperones.
  • especially preferred "essential" chaperones are yeast chaperones. including chaperones comprising the sequence of proteins encoded by a gene selected from CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNSl, EROl (in the absence of diamide), HSPlO., HSP 60, PDIl, CDCSl, KAR2, MGEl, MRSIl, NOBl, SSCl, PSEl, TIM9, PAMl 8 and TCPl.
  • a host cell that is mutated to inactive EROl can be complemented by growth in the presence of the oxidant diamide (Frand & Kaiser, 1998, Molecular Cell, I 5 161-170), but diamide is not a "nutrient" addition of the type discussed above in respect of auxotrophic mutations.
  • Diamide is not a commonly used component of growth media and an EROl mutant that is transformed with a plasmid comprising the EROl gene can be grown in rich media without loss of plasmid stability.
  • the present invention also provides a host cell comprising a plasmid (such as a plasmid as defined above by any of the previous aspects of the invention), the plasmid comprising a gene that encodes an
  • essential protein such as a chaperone
  • the host cell in the absence of the plasmid, the host cell is unable to produce the "essential" protein.
  • the host cell is inviable.
  • the host cell has been genetically modified to render it unable to produce a functional copy of the
  • the host cell may, or may not, further comprise a recombinant gene encoding a heterologous protein, such as those described above in respect of earlier aspects of the invention.
  • the present invention also provides, in a twelfth aspect, a plasmid comprising, as the sole yeast selectable marker, optionally as the sole selectable marker, a gene encoding an "essential" protein, such as an ''essential" chaperone.
  • the plasmid may. or may not, further comprise a gene encoding a heterologous protein.
  • the plasmid may, or may not be a 2 ⁇ m-family plasmid.
  • the present invention also provides, in a thirteenth aspect, a method for producing a desired protein (such as a desired heterologous protein) comprising the steps of: providing a host cell comprising a plasmid, the plasmid comprising a gene that encodes an "essential" protein, such as a chaperone, wherein, in the absence of the plasmid, the host cell is unable to produce the "essential" protein and wherein the host cell further comprises a gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein); culturing the host cell in a culture medium under conditions that allow the expression of the "essential" protein and the desired protein; and optionally purifying the thus expressed desired protein from the cultured host cell or the culture medium: and further optionally, lyophilising the thus purified protein.
  • a desired protein such as a desired heterologous protein
  • a host cell used in this method may, or may not, be a host cell according to the eleventh aspect of the invention and/or the host call may, or may not, be transformed with a plasmid according to the twelfth aspect of the invention.
  • the method may, or may not, further comprise the step of formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form, in the manner discussed above.
  • the purified desired protein such as a desired heterologous protein
  • the step of "culturing the host cell in a culture medium under conditions that allow the expression of the "essential" protein and the desired protein” involves culturing the host cell in medium that is not specifically adapted to be selective for the presence of any genes on the plasmid, other than for the presence of the gene encoding the "essential" protein.
  • the step of culturing the host cells may. or may not. be performed in non-selective media, such as complex or rich media and/or under conditions (such as pH, temperature and/or oxygen levels) that are not specifically adapted to select for the presence of the '"essential" protein.
  • a medium can be described as non-selective for the purposes of the present situation if it is not specifically adapted to deprive the host cell of a product, typically a nutrient product, that is ordinarily provided to maximise, or otherwise allow, the growth of host cells that have not been modified to prevent the expression of the "essential" protein.
  • a medium may be a non-selective medium, for the purposes of the present invention if, when one compares plasmid stability in a first host cell type grown in the 'lest medium” to plasmid stability in a second cell type grown in the "test medium” when each cell type is grown for 5, 10, 15, 20, 25 or 30 generations in the "test medium", wherein - (i) the first host cell type is a host cell according to the eleventh aspect of the present invention;
  • the second host cell type is a host cell according to the eleventh aspect of the present invention except that it has been modified to restore the ability of the host cell to produce the "essential" protein in the absence of the plasmid (which is not to say that the second host cell type does not contain a plasmid encoding the "essential” protein, just that it can produce the "essential” protein even when the plasmid is not present); then the plasmid stability observed in the second cell type is less than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of the plasmid stability observed in the first cell type.
  • a fourteenth aspect of the present invention also provides for the use of a polynucleotide that encodes an "essential" protein (as defined above) to increase the stability of a plasmid in a host cell, particularly under non-selective conditions, by integration of the polynucleotide into the plasmid to produce a modified plasmid. wherein the host cell is unable to produce the '"essential" protein in the absence of the modified plasmid.
  • the increase in stability may. or may not.
  • the plasmid may, or may not, additionally comprise a gene that encodes a further desired heterologous protein, such as defined above in respect of earlier aspects of the present invention.
  • the use may, or may not, be to improve the productivity of the desired heterologous protein, such as when the host cell comprising the plasmid is grown under non-selective conditions.
  • the "essential" protein is a chaperone and may, or may not, be used to simultaneously increase the stability of a plasmid in the host cell and increase the ability of the host cell to produce a desired protein product.
  • the desired protein product may, or may not, be an endogenous! ⁇ ' encoded protein or may, or may not, be a heterologous protein as defined by the earlier aspects of the invention.
  • the protein product is a heterologous protein, it may, or may not, be encoded by a recombinant gene that has been integrated into the chromosome of the host cell, or by a gene that is present on a plasmid in the host cell, such as the modified plasmid comprising the polynucleotide that encodes the "essential" protein as defined above.
  • a recombinant gene that has been integrated into the chromosome of the host cell, or by a gene that is present on a plasmid in the host cell, such as the modified plasmid comprising the polynucleotide that encodes the "essential" protein as defined above.
  • the effects of recombinantly-provided chaperones according to the other embodiments of the present invention can be modulated by modifying the promoters that control the expression levels of the chaperone(s). Surprisingly we have found that, in some cases, shorter promoters result in increased expression of a desired protein.
  • a polynucleotide such as a plasmid as defined above
  • a promoter operably connected to a coding sequence encoding a chaperone (such as those described above), for use in increasing the expression of a desired protein (such as a desired heterologous protein), such as those described above, in a host cell (such as those described above) by expression of the polynucleotide sequence within the host cell, wherein the promoter is characterised in that it achieves a modified, such as a higher or lower, level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter.
  • the present invention also provides, in a sixteenth aspect, a method for producing a desired protein (such as a desired heterologous protein) comprising the steps of: providing a host cell comprising a recombinant gene that comprising the sequence of promoter operably connected to a coding sequence encoding a chaperone, the promoter being characterised in that it achieves a lower level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter, and the host cell further comprising a gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein); culturing the host cell under conditions that allow the expression of the chaperone and the desired protein; and optionally purifying the thus expressed desired protein from the cultured host cell or the culture medium; and further optionally, lyophilising the thus purified protein; and optionally further formulating the purified desired protein with a carrier or diluent; and optionally presenting the thus
  • the combination of recombinantly expressed PDI and transferrin-based proteins provides a surprisingly high level of transferrin expression.
  • transferrin expression in a system that includes a chromosomally encoded recombinant PDI gene provided a 2-fold increase (compared to a control in which there is no chromosomally encoded recombinant PDI gene). This increase was 5-times greater than an equivalent system comprising a recombinant gene encoding human albumin in place of the recombinant transferrin gene.
  • the host may be any cell type, such as a procaryotic cell (e.g. bacterial cells such as E. col ⁇ ) or a eucaryotic cell.
  • a procaryotic cell e.g. bacterial cells such as E. col ⁇
  • a eucaryotic cell e.g. bacterial cells such as E. col ⁇
  • Preferred eucaryotic cells include fungal cells, such as yeast cells, and mammalian cells. Exemplar ⁇ 7 yeast cells are discussed above. Exemplary mammalian cells include human cells.
  • Host cells as described above can be cultured to produce recombinant transferrin- based proteins.
  • the thus produced transferrin-based proteins can be isolated from the culture and purified, optionally to a pharmaceutically acceptable level of purity, for example using techniques known in the art and/or as set out above.
  • Purified transferrin-based proteins may, or may not, be formulated with a pharmaceutically acceptable carrier or diluent and may, or may not, be presented in unit dosage form.
  • Figure 1 shows a map of a typical 2 ⁇ m plasmid.
  • Figure 2 shows the results of rocket immunoelectrophoresis (RIE) determination of increased recombinant transferrin (N413Q, N61 IQ) secretion with PDIl over- expression.
  • Cryopreserved yeast stocks were grown for 4-days in 1OmL BMMD shake flask cultures and supernatants were loaded at 5 ⁇ L per well.
  • Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) was used at 40 ⁇ L per rocket immunoelectrophoresis gel (5OmL).
  • Figure 3 shows the results of RIE analysis of recombinant transferrin (N413Q, N611Q) secretion with and without PDIl over-expression.
  • Cryopreserved yeast stocks were grown for 4-days in 1OmL BMMD shake flask cultures and supernatants were loaded at 5 ⁇ L per well. Duplicate loadings were made of supernatants from two individual cultures of each strain. Goat polyclonal anti- transferrin (human) antiserum (Calbiochem) was used at 40 ⁇ L per rocket immunoelectrophoresis gel (5OmL).
  • Figure 4 shows the results of SDS-PAGE analysis of recombinant transferrin secretion with and without PDIl over-expression.
  • BMMD shake flask cultures were grown for 4-days and 10 ⁇ L supernatant analysed on non-reducing SDS- PAGE (4-12% NuP AGE ® , MOPS buffer, inVitrogen) with GelCode ® Blue reagent (Pierce).
  • 1 SeeBlue Plus2 Markers (InVitrogen).
  • Figure 5 shows RJE analysis of recombinant transferrin secretion from S. cerevisiae strains with an additional integrated copy of PDIl .
  • 5-day BMMD shake flask culture supernatants were loaded at 5mL per well.
  • Strains contained: 1) pSAC35 (negative control); 2) pDB2536 (recombinant non-glycosylated transferrin (N413Q, N611Q)) or 3) pDB2506 (same as plasmid pDB2536 but the transferrin ORF encodes transferrin without the N ⁇ Q mutations at positions 413 and 611, i.e. recombinant glycosylated transferrin).
  • Each well contained a sample derived from an individual transformant.
  • Standards were human plasma holo- transferrin (Calbiochem) at 100, 50, 20, 10, 5 and 2mg.l ⁇
  • Figure 6 shows RIE analysis of recombinant transferrin secretion from Strain A [pDB2536] and Strain A [pDB2506] grown in shake flask culture. 5-day BMMD or YEPD shake flask culture supernatants were loaded in duplicate at 5mL per well.
  • Figure 7 shows SDS-PAGE analysis of recombinant transferrin secreted from Strain A [pDB2536] and Strain A [pDB2506] grown in shake flask culture. Cultures were grown for 5 -days in BMMD and 3OmL supernatants analysed on SDS-PAGE (4-12% NuP AGETM, MOPS Buffer, InVitrogen) stained with GelCode, Blue Reagent (Pierce).
  • Figure 8 shows RIE of recombinant transferrin secreted from 5 cerevisiae strains with different PDIl copy numbers. 3 -day BMMD shake flask culture supernatants were loaded at 5mL per well. Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) was used at 3OmL per rocket Immunoelectrophoresis gel (5OmL).
  • Figure 9 shows SDS-PAGE analysis of recombinant transferrin secreted from S. cerevisiae strains with different PDIl copy numbers. 4-12% NuPAGE reducing gel run with MOPS buffer (InVitrogen) after loading with 3OmL of 3-day BMMD shake flask culture supernatant per lane; (lane 1) supernatant from control strain [pDB2536]; (lane 2) supernatant from Strain A [pDB2536]; (lanes 3-6) supernatant from control strain [pDB271 1] or [pDB2712]; (lane 7) molecular weight markers (SeeBlue Plus2. InVitrogen).
  • MOPS buffer InVitrogen
  • Figure 10 shows RIE of recombinant transferrin secreted from different S. cerevisiae strains with and without additional PDIl gene co-expression.
  • 1OmL YEPD shake flasks were inoculated with yeast and incubated for 4-days at 30°C. 5 ⁇ L culture supernatant loaded per well of a rocket Immunoelectrophoresis gel.
  • Plasma Tf standards concentrations are in ⁇ g/mL. 20 ⁇ L goat anti-Tf / 5OmL agragose. Precipin was stained with Coomassie blue.
  • Figure 11 shows RIE analysis of rHA expression in different S. cerevisiae strains when co-expressed with PDIl genes having different length promoters. 1OmL
  • YEPD shake flasks were inoculated with yeast and incubated for 4-days at 30°C.
  • FIG. 12 shows RIE analysis of rHA expression in different S. cerevisiae strains when co-expressed with PDIl genes having different length promoters. 1OmL YEPD shake flasks were inoculated with yeast and incubated for 4-days at 30 0 C. 4 ⁇ L culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in ⁇ g/mL. 400 ⁇ L goat anti-HA (Sigma product A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue.
  • Figure 13 shows RIE analysis of rHA fusion proteins with and without co- expressed recombinant PDIl.
  • 1OmL BMMD shake flasks were inoculated with YBX7 transformed with albumin fusion expression plasmids and incubated for 4- days at 30 0 C. 4 ⁇ L culture supernatant loaded per well of a rocket immunoelectrophoresis gel.
  • rHA standards concentrations are in ⁇ g/mL.
  • 200 ⁇ L goat anti-HA Sigma product A-1151 resuspended in 5mL water
  • Precipin was stained with Coomassie blue.
  • Figure 14 shows SDS-PAGE analysis of recombinant albumin fusion secretion with and without PDIl present on the expression plasmid.
  • 1OmL BMMD shake flasks were inoculated with yeast and incubated for 4-days at 30 0 C. 200rpm.
  • 30 ⁇ L supernatant analysed on non-reducing SDS-PAGE (4-12% NuPAGE ® , MES buffer, InVitrogen) with GelCode ® Blue reagent (Pierce).
  • FIG. 15 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with 0RM2 co-expression from a 2 ⁇ m-based plasmid.
  • Four day shake flask culture supernantants were loaded at 5 ⁇ l per well.
  • Standards were human plasma holo-transferrin (Calbiochem), at 25, 20, 15. 10, 5 ⁇ g/ml, loaded 5 ⁇ l per well.
  • Goat polyclonal anti -transferrin (human) antiserum (Calbiochem) used at 20 ⁇ l per rocket immunoelectrophoresis gel (50 ml).
  • Figure 16 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with PSEl co-expression from a 2 ⁇ m-based plasmid.
  • Four day shake flask culture supernantants were loaded at 5 ⁇ l per well.
  • Standards were human plasma holo-transferrin (Calbiochem), at 25. 20, 15, 10. 5 ⁇ g/ml, loaded 5 ⁇ l per well.
  • Figure 17 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with SSAl co-expression from a 2 ⁇ m-based plasmid.
  • Four day shake flask culture supernantants were loaded at 5 ⁇ l per well.
  • Standards were human plasma holo-transferrin (Calbiochem). at 25, 20, 15, 10, 5 ⁇ g/ml, loaded 5 ⁇ l per well.
  • Figure 18 shows the results of RIE.
  • 1OmL YEPD shake flasks were inoculated with DXYl trpl ⁇ [pDB2976], DXYl trpl ⁇ [pDB2977], DXYl trpl ⁇ [pDB2978], DXYl trpl ⁇ [pDB2979], DXYl trpl ⁇ [pDB2980] or DXYl trpl ⁇ [pDB2981] transformed to tryptophan prototrophy with a 1.41kb NotVPstl pdiL.
  • TRPl disrupting DNA fragment was isolated from pDB3078. Transformants were grown for 4-days at 30 0 C, 200rpm.
  • Figure 19 shows the results of RIE.
  • 1OmL YEPD shake flasks were inoculated with DXYl [pDB2244], DXYl [pDB2976], DXYl trpl ⁇ pdil::TRPl [pDB2976], DXYl [pDB2978], DXYl trpl ⁇ pdilr.
  • Figure 20 shows a sequence alignment of the SKQ2n and S288c gene sequences with long promoters, as described in Example 6.
  • Figures 21 to 33 show various plasmid maps.
  • Figure 34 shows Rocket Immunoelectrophoresis of YEPD shake flask culture supernatants from DXYl and DXYl ⁇ trpl pdilrTRPl containing pDB3175 to pDB3178.
  • 1OmL YEPD shake flasks were inoculated with DXYl [pAYE316], DXYl [pDB3175], DXYl [pDB3176], DXYl [pDB3177], DXYl [pDB3178], DXYl ⁇ trpl pdilrTRPl [pDB3175], DXYl ⁇ trpl pdilr TRPl [pDB3176], DXYl ⁇ trpl pdilr.
  • TRPl [pDB3177], and DXYl ⁇ trpl pdilrTRPl [pDB3178] were grown for 4-days at 3O°C. 200rpm.
  • 5 ⁇ L culture supernatant was loaded per well of a 5OmL rocket immunoelectrophoresis gel in triplicate.
  • rHA standard concentrations were 300, 200 : 150, 100 and 50 ⁇ g/mL.
  • Precipitin was stained with Coomassie blue.
  • Figure 35 shows Rocket Immunoelectrophoresis of YEPD shake flask culture supernatants from DXYl and DXYl Atrpl pdil::TRPl containing pDB3179 to pDB3182.
  • 1OmL YEPD shake flasks were inoculated with DXYl [pDB2931], DXYl [pDB3179], DXYl [pDB3180], DXYl [pDB3181], DXYl [pDB3182], DXYl ⁇ trpl pdil::TRPl [pDB3179], DXYl Atrpl pdilr.
  • TRPl [pDB3180], DXYl Atrpl pdilr. TRPl [pDB3181], and DXYl Atrpl pdil::TRPl [pDB3182] were grown for 4-days at 30°C, 200rpm. 5 ⁇ L culture supernatant was loaded per well of a 5OmL rocket immunoelectrophoresis gel in triplicate. Plasma transferrin standard concentrations were 100, 50, 20, 10 and 5 ⁇ g/mL. 40 ⁇ L goat polyclonal anti human transferrin antiserum (Calbiochem) was used per 5OmL agarose gel. Precipitin was stained with Coomassie blue.
  • Two types of expression cassette ha ⁇ 'e been used to exemplify secretion of a recombinant human transferrin mutant (N413Q, N611Q) from S. cerevisiae.
  • One type uses a modified HSA(pre)/MF ⁇ l(pro) leader sequence (named the "modified fusion leader” sequence).
  • the second type of expression cassette uses only the modified HSA(pre) leader sequence.
  • the 24 amino acid sequence of the "modified fusion leader” is MKWVFIVSILFLFSSAYSRSLDKR.
  • the 18 amino acid sequence of the modified HSA(pre) leader sequence is MKWVFIVSILFLFSSAYS. Transferrin (N413Q, N611Q) expression using these two cassettes has been studied in S. cerevisiae using the 2 ⁇ m expression vector with and without an additional copy of the S. cerevisiae PDl gene. PDIl .
  • Plasmids pDB2515, pDB2529, pDB2536, pDB2688, pDB2690, pDB2711, pDB2921, pDB2928, pDB2929, pDB2930, pDB293L pDB2932 and pDB2690 were constructed as described in Example 1 of WO 2005/061718. the contents of which are incorporated herein by reference.
  • a S. cerevisiae control strain was transformed to leucine prototrophy with all the transferrin (N413Q, N611Q) expression plasmids, and cryopreserved stocks were prepared.
  • Figure 4 shows an SDS-PAGE comparison of the recombinant transferrin secreted by S. cerevisiae strains with and without additional PDIl expression.
  • S. cerevisiae Strain A was selected to investigate the secretion of recombinant glycosylated transferrin expression from plasmid pDB2506 and recombinant non- glycosylated transferrin (N413Q, N61 IQ) from plasmid pDB2536.
  • Strain A has the following characteristics -
  • a control strain had none of the above insertions.
  • Control strain [cir°] and Strain A [cir°] were transformed to leucine prototrophy with pDB2506 (recombinant transferrin), pDB2536 (recombinant non- glycosylated transferrin (N413Q, N611Q)) or pSAC35 (control). Transformants were selected on BMMD-agar.
  • Plasmid pDB2711 is as described above. Plasmid pDB2712 ( Figure 22 of WO 2005/061718) was also produced with the Notl cassette in the opposite direction to pDB2711.
  • Control strain S. cerevisiae [cir°] was transformed to leucine prototrophy with pDB2711 and pDB2712. Transformants were selected on BMMD-agar and cryopreserved trehalose stocks of control strain [pDB2711] were prepared.
  • Control strain [pDB2711] and control strain [pDB2712] appeared to secrete similar levels of rTf (N413Q, N61 IQ) into the culture media.
  • the levels of secretion were relatively consistent between control strain [pDB2711] and control strain [pDB2712] transformants in both BMMD and YEPD media, suggesting that plasmid stability was sufficient for high-level transferrin secretion even under non-selective conditions. This is m contrast to the previous published data in relation to recombinant PDGF-BB and HSA where introduction of PDIl into multicopy 2 ⁇ m plasmids was shown to be detrimental to the host.
  • the steady state PDIl mRNA level could be increased by either increasing the transcription rate, say by use of a higher efficiency promoter, or by reducing the clearance rate of the PDIl mRNA.
  • protein engineering could be used to enhance the specific activity or turnover number of the Pdilp protein.
  • control strain [pDB2711] recombinant transferrin (N413Q, N61 IQ) production was measured at approximately 3g.L " ' by both GP-HPLC analysis and SDS-PAGE analysis (Table 2). This level of production is several fold-higher than control strain, the alternative control strain or Strain A containing pDB2536. Furthermore, for the production of proteins for therapeutic use in humans, expression systems such as control strain [pDB2711] have advantages over those using Strain A, as they do not contain bacterial DNA sequences.
  • N611Q at an elevated level compared to strains containing a single copy of PDIl .
  • a further increase in PDIl copy number was achieved by using pDB271 1
  • recombinant transferrin N413Q, N611Q
  • Control strain [pDB2711] which contained,the PDIl gene on the multicopy expression plasmid, produced a several-fold increase in recombinant transferrin (N413Q, N611Q) secretion compared to Strain A [pDB2536] in both shake flask culture and high cell density fermentation.
  • Elevated PDIl copy number in yeast such as S. cerevisiae will be advantageous during the production of desired proteins (such as a desired heterologous proteins), such as those from the transferrin family.
  • desired proteins such as a desired heterologous proteins
  • pSAC35-based plasmids containing additional copies of PDIl gene have advantages for the production of proteins from the transferrin family, and their derivatives, such as fusions, mutants, domains and truncated forms.
  • the S. cerevisiae strain JRYl 88 cir + (National Collection of Yeast Cultures) and MT302/28B cir + (Finnis et al, 1993, Eur. J. Biochem., 212, 201-210) was cured of the native 2 ⁇ m plasmid by galactose induced over-expression of FLP from Ye ⁇ 351 -GAL-FLPl, as described by Rose and Broach (1990, Meth. EnzymoL, 185, 234-279) to create the S. cerevisiae strains JRYl 88 cir 0 and MT302/28B cir°, respectively.
  • the PDIl genes from S. cerevisiae S288c and S. cerevisiae SKQ2n were amplified by PCR to produce DNA fragments with different lengths of the 5'- untranslated region containing the promoter sequence.
  • PCR primers were designed to permit cloning of the PCR products into the £coRI and BamHl sites of YIplac211 (Gietz & Sugino, 1988, Gene, 74, 527-534). Additional restriction endonuclease sites were also incorporated into PCR primers to facilitate subsequent cloning.
  • Table 3 describes the plasmids constructed and Table 4 gives the PCR primer sequences used to amplify the PDIl genes. Differences in the PDIl promoter length within these YIplac211 -based plasmids are described in Table 3.
  • pDB2939 ( Figure 27 of WO 2005/061718) was produced by PCR amplification of the PDIl gene from 5. cerevisiae S288c genomic DNA with oligonucleotide primers DS248 and DS250 (Table 5), followed by digesting the PCR product with EcoKL and Bam ⁇ l and cloning the approximately 1.98-kb fragment into YIplac211 (Gietz & Sugino, 1988, Gene, 74, 527-534), that had been cut with £c ⁇ RI and BaniHl. DNA sequencing of pDB2939 identified a missing 'G' from within the DS248 sequence, which is marked in bold in Table 4.
  • Oligonucleotide primers used for sequencing the PDIl gene are listed in Table 5, and were designed from the published S288c PDIl gene sequence (PD11/YCL043C on chromosome III from coordinates 50221 to 48653 plus 1000 base pairs of upstream sequence and 1000 base pairs of downstream sequence. (http://wwvy.veastgenorne.org/ Genebank Accession number NCOOl 135).
  • Plasmids pDB2941 ( Figure 28 of WO 2005/061718) and pDB2942 ( Figure 29 of WO 2005/061718) were constructed similarly using the PCR primers described in Tables 3 and 4, and by cloning the approximately 1.90-kb and 1.85-kb EcoRl- BamEl fragments, respectively, into YIplac211. The correct DNA sequences were confirmed for the PDU genes in pDB2941 and pDB2942. The S.
  • SKQ2n PDIl gene sequence was PCR amplified from plasmid DNA containing the PDIl gene from pMA3a:C7 (US 6 : 291,205) ; also known as Clone C7 (Crouzet & Tuite, 1987, supra; Farquhar et al, 1991. supra).
  • the SKQ2n PDU gene was amplified using oligonucleotide primers DS248 and DS250 (Tables 3 and 4).
  • the approximately 2.01-kb PCR product was digested with EcoRl and BamHl and ligated into YIplac21 1 (Gietz & Sugino, 1988, Gene, 74, 527-534) that has been cut with EcoKL and BamHl, to produce plasmid pDB2943 ( Figure 30 of WO 2005/061718).
  • the 5 1 end of the SKQ2n PDIl sequence is analogous to a blunt-ended Spel- ⁇ te extended to include the EcoBl, Sad, SnaBl, Pad, Fsel, Sfil and Smal sites, the 3' end extends up to a site analogous to a blunt-ended Bsu36l site, extended to include a Smal, SnaBl and BamHl sites.
  • the PDIl promoter length is approximately 210bp.
  • the entire DNA sequence was determined for the PDIl fragment using oligonucleotide primers given in Table 5. This confirmed the presence of a coding sequence for the PDI protein of S.
  • Plasmids pDB2963 ( Figure 31 of WO 2005/061718) and pDB2945 ( Figure 32 of WO 2005/061718) were constructed similarly using the PCR primers described in Tables 3 and 4, and by cloning the approximately 1.94-kb and 1.87-kb EcoRI- BamHl fragments, respectively, into YIplac211. The expected DNA sequences were confirmed for the PDIl genes in pDB2963 and pDB2945, with a serine codon at the position of amino acid 114.
  • pSAC35-based plasmids were constructed for the co-expression of rHA with different PDIl genes (Table 6).
  • pDB2693 was digested with SnaBl, treated with calf intestinal alkaline phosphatase, and ligated with SnciBl fragments containing the PDIl genes from pDB2943, ⁇ DB2963, pDB2945 ; pDB2939, pDB2941 and pDB2942. This produced plasmids pDB2976 to pDB2987 ( Figures 35 to 46 of WO 2005/061718). PDIl transcribed in the same orientation as REP2 was designated “orientation A", whereas PDIl transcribed in opposite orientation to REP2 was designated “orientation B" (Table 6).
  • pSAC35-based plasmids were constructed for the co-expression of recombinant transferrin (N413Q, N61 IQ) with different PDIJ genes (Table 7).
  • Table 7 pSAC35-based plasmids for co-expression of transferrin with different PDIl genes
  • Notl expression cassettes for rHA expression were first deleted from pDB2976, pDB2978, and pDB2980 by Notl digestion and circularisation of the vector backbone. This produced plasmids pDB3081 ( Figure 47 of WO 2005/061718), pDB3083 ( Figure 48 of WO 2005/061718) and pDB3084 ( Figure 49 of WO 2005/061718) as described in Table 8.
  • Table 8 pSAC35-based plasmids with different PDIl genes
  • the albumin titre in the culture supernatants in the absence of PDIl on the plasmid was dependant upon which yeast strain was selected as the expression host, however, in most examples tested the largest increase in expression was observed when PDIl with the long promoter ( ⁇ 210-bp) was present in the 2 ⁇ m plasmid (pDB2976). Modifying the PDIl promoter by shortening, for example to delete regulation sites, had the affect of controlling the improvement. For one yeast strain, known to be a high rHA producing strain (DS569) a shorter promoter was preferred for optimal expression.
  • PDIl on the 2 ⁇ m-based plasmid enhanced the secretion of recombinant alb umin fusions.
  • Table 9 Summary of plasmids encoding albumin fusion proteins
  • the S". cerevisiae Control Strain and Strain A were selected to investigate the effect on transferrin secretion when the transferrin and 0RM2 genes were co-expressed from the 2 ⁇ m -based plasmids.
  • the Control Strain and Strain A were transformed to leucine prototrophy by plasmids pDB3090, pDB3092 and pBD3093 (containing expression cassettes for rTf (N413Q, N611Q) and for 0RM2), as well as a control plasmid pDB2931 (containing the rTf (N413Q, N611Q) expression cassette without 0RM2).
  • the construction of these plasmids is described in Example 10 of WO 2005/061718, the contents of which are incorporated herein by reference. Transformants were selected on BMMD agar and patched out on BMMD agar for subsequent analysis.
  • the S. cerevisiae Control Strain was transformed to leucine prototrophy by plasmids, pDB3097 and pBD3098 (containing expression cassettes for rTf
  • the S. cerevisiae Control Strain was transformed to leucine prototrophy by plasmids, pDB3094 and pBD3095 (containing expression cassettes for rTf (N413Q, N61 1Q) and for SSAl), as well as a control plasmid pDB2931 (containing the rTf (N413Q, N61 1 Q) expression cassette without SSAl).
  • plasmids containing expression cassettes for rTf (N413Q, N61 1Q) and for SSAl
  • a control plasmid pDB2931 containing the rTf (N413Q, N61 1 Q) expression cassette without SSAl.
  • Transformants were selected on BMMD agar and patched out on BMMD agar for subsequent analysis.
  • PDIl gene disruption combined with a PDIl gene on the 2 ⁇ m-based plasmid enhanced the secretion of recombinant albumin and plasmid stability.
  • Single stranded oligonucleotide DNA primers listed in Table 11 were designed to amplify a region upstream of the yeast PDIl coding region and another a region downstream of the yeast PDIl coding region.
  • Primers DS299 and DS300 amplified the 5' region of PDIl by PCR, while primers DS301 and DS302 amplified a region 3' of PDIl, using genomic DNA derived S288c as a template.
  • PCR conditions were as follows: l ⁇ L S288c template DNA (at O.Olng/ ⁇ L, O.lng/ ⁇ L, lng/ ⁇ L, lOng/ ⁇ L and lOOng/ ⁇ L), 5 ⁇ L lOXBuffer (Fast Start Taq+Mg, (Roche)), l ⁇ L 1OmM dNTP's, 5 ⁇ L each primer (2 ⁇ M), 0.4 ⁇ L Fast Start Taq, made up to 50 ⁇ L with H 2 O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9700.
  • the conditions were: denature at 95°C for 4min [HOLD], then [CYCLE] denature at 95°C for 30 seconds, anneal at 45°C for 30 seconds, extend at 72°C for 45 seconds for 20 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C.
  • the 0.22kbp PDIl 5' PCR product was cut with Notl and HmdIIL while the 0.34kbp PDIl 3' PCR product was cut with HmdIII and P stl.
  • Plasmid pMCS5 (HoheiseL 1994, Biotechniques 17, 456-460) ( Figure 85 of WO 2005/061718) was digested to completion with Hin ⁇ lll, blunt ended with T4 DNA polymerase plus dNTPs and religated to create pDB2964 ( Figure 86 of WO 2005/061718).
  • Plasmid pDB2964 was Hindl ⁇ i digested, treated with calf intestinal phosphatase, and ligated with the 0.22kbp PDIl 5' PCR product digested with Notl and HindHl and the 0.34kbp PDIl 3' PCR product digested with Hin ⁇ lll and PsA to create pDB3069 ( Figure 87 of WO 2005/061718) which was sequenced with forward and reverse universal primers and the DNA sequencing primers DS303, DS304, DS305 and DS306 (Table 11).
  • Primers DS234 and DS235 were used to amplify the modified TRPl marker gene from Ylplac204 (Gietz & Sugino, 1988, Gene, 74, 527-534), incorporating Hindl ⁇ l restriction sites at either end of the PCR product.
  • PCR conditions were as follows: l ⁇ L template YIplac204 (at O.Olng/ ⁇ L, O.lng/ ⁇ L, lng/ ⁇ L, lOng/ ⁇ L and lOOng/ ⁇ L), 5 ⁇ L lOXBuffer (Fast Start Taq+Mg, (Roche)), l ⁇ L 1OmM dNTP's, 5 ⁇ L each primer (2 ⁇ M), 0.4 ⁇ L Fast Start Taq, made up to 50 ⁇ L with H 2 O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9600.
  • the 0.86kbp TRPl gene was isolated from pDB2778 by digestion with Hin ⁇ lll and cloned into the Hindlll site of pDB3069 to create pDB3078 ( Figure 89 of WO 2005/061718) and pDB3079 ( Figure 90 of WO 2005/061718).
  • a 1.41kb pdil :TRPl disrupting DNA fragment was isolated from pDB3078 or pDB3079 by digestion with NotllPstl.
  • Yeast strains incorporating a TRPl deletion were to be constructed in such a way that no homology to the TRPl marker gene (pDB2778) should left in the genome once the trpl ⁇ had been created, so preventing homologous recombination between future TRPl containing constructs and the TRPl locus.
  • oligonucleotides were designed to amplify areas of the 5' UTR and 3' UTR of the TRPl gene outside of TRPl marker gene present on integrating vector YIplac204 (Gietz & Sugino, 1988, Gene, 74, 527-534).
  • the YIplac204 TRPl marker gene differs from the native/chromosomal TRPl gene in that internal Hzndlll, Pstl and Xbal sites were removed by site directed mutagenesis (Gietz & Sugino, 1988, Gene, 74, 527-534).
  • the YIplac204 modified TRPl marker gene was constructed from a 1.453kbp blunt-ended genomic fragment EcoR ⁇ fragment, which contained the TRPl gene and only 102bp of the TRPl promoter (Gietz & Sugino, 1988, Gene, 74, 527-534).
  • Single stranded oligonucleotide DNA primers were designed and constructed to amplify the 5' UTR and 3' UTR regions of the TRPl gene so that during the PCR amplification restriction enzyme sites would be added to the ends of the PCR products to be used in later cloning steps.
  • Primers DS230 and DS231 (Table 12) amplified the 5' region of TRPl by PCR, while primers DS232 and DS233 (Table 12) amplified a region 3' of TRPl, using S288c genomic DNA as a template.
  • PCR conditions were as follows: l ⁇ L template S288c genomic DNA (at O.Olng/ ⁇ L, O.lng/ ⁇ L, lng/ ⁇ L, lOng/ ⁇ L and lOOng/ ⁇ L), 5 ⁇ L lOXBuffer (Fast Start Taq+Mg ; (Roche)), l ⁇ L 1 OmM dNTP's, 5 ⁇ L each primer (2 ⁇ M), 0.4 ⁇ L Fast Start Taq, made up to 50 ⁇ L with H 2 O. PCRs were performed using a Perkin- Elmer Thermal Cycler 9600.
  • the 0.19kbp TRPl 5' UTR PCR product was cut with Ec ⁇ Rl and Hin ⁇ lll, while the 0.2kbp TRPl 3' UTR PCR product was cut with BamUl and Hindlll and ligated into pAYE505 linearised with BamHl/EcoRl to create plasmid pDB2777 ( Figure 91 of WO 2005/061718).
  • the construction of pAYE505 is described in WO 95/33833 .
  • DNA sequencing using forward and reverse primers, designed to prime from the plasmid backbone and sequence the cloned inserts confirmed that in both cases the cloned 5' and 3' UTR sequences of the TRPl gene had the expected DNA sequence.
  • Plasmid pDB2777 contained a TRPl disrupting fragment that comprised a fusion of sequences derived from the 5' and 3' UTRs of TRPl. This 0.383kbp TRPl disrupting fragment was excised from pDB2777 by complete digestion with EcoRl.
  • Yeast strain DXYl (Kerry- Williams et al, 1998, Yeast, 14, 161-169) was transformed to leucine prototrophy with the albumin expression plasmid pDB2244 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST- 1, protocol 2; (Ito et al, 1983, J. BacterioL, 153, 163; Elble, 1992, Biotechniques, 13, 18)) to create yeast strain DXYl [pDB2244].
  • the construction of the albumin expression plasmid pDB2244 is described in WO 00/44772. Transformants were selected on BMMD-agar plates, and were subsequently patched out on BMMD- agar plates.
  • Cryopreserved trehalose stocks were prepared from 1OmL BMMD shake flask cultures (24 hrs, 3O°C, 200rpm).
  • DXYl [pDB2244] was transformed to tryptophan autotrophy with the 0.383kbp EcoRl TRPl disrupting DNA fragment from pDB2777 using a nutrient agar incorporating the counter selective tryptophan analogue.
  • 5-fluoroanthranilic acid 5-FAA
  • Colonies resistant to the toxic effects of 5-FAA were picked and streaked onto a second round of 5-FAA plates to confirm that they really were resistant to 5-FAA and to select away from any background growth. Those colonies which grew were then were re-patched onto BMMD and BMMD plus tryptophan to identify which were tryptophan auxotrophs.
  • PCR amplification across the TRPl locus was used to confirm that the trp " phenotype was due to a deletion in this region.
  • Genomic DNA was prepared from isolates identified as resistant to 5-FAA and unable to grow on minimal media without the addition of tryptophan.
  • PCR amplification of the genomic TRPl locus with primers CED005 and CED006 was achieved as follows: l ⁇ L template genomic DNA, 5 ⁇ L lOXBuffer (Fast Start Taq+Mg, (Roche)), l ⁇ L 1OmM dNTP's, 5 ⁇ L each primer (2 ⁇ M), 0.4 ⁇ L Fast Start Taq, made up to 50 ⁇ L with H 2 O.
  • PCRs were performed using a Perkin-Elmer Thermal Cycler 9600. The conditions were: denature at 94°C for lOmin [HOLD], then [CYCLE] denature at 94°C for 30 seconds, anneal for 30 seconds at 55°C, extend at 72°C for 120sec for 40 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C.
  • PCR amplification of the wild type TRPl locus resulted in a PCR product of 1.34kbp in size, whereas amplification across the deleted TRPl region resulted in a PCR product 0.84kbp smaller at 0.50kbp.
  • PCR analysis identified a DXYl derived trp " strain (DXYl trpl A [pDB2244]) as having the expected deletion event.
  • the yeast strain DXYl trpl ⁇ [pDB2244] was cured of the expression plasmid pDB2244 as described by Sleep et ah, (1991, Bio/Technology, 9, 183-187).
  • DXYl trplA cir 0 was re-transformed the leucine prototrophy with either pDB2244, pDB2976, pDB2977, pDB2978, pDB2979, pDB2980 or pDB2981 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST- I 5 protocol 2; (Ito et al, 1983, J Bacteriol, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD-agar plates supplemented with tryptophan, and were subsequently patched out on BMMD-agar plates supplemented with tryptophan. Cryopreserved trehalose stocks were prepared from 1OmL BMMD shake flask cultures supplemented with tryptophan (24 hrs, 3O°C, 200rpm).
  • the yeast strains DXYl trpl ⁇ [pDB2976], DXYl trpl ⁇ [pDB2977], DXYl trplA [pDB2978], DXYl trpl ⁇ [pDB2979], DXYl trpl ⁇ [pDB2980] or DXYl trpl ⁇ [pDB2981] was transformed to tryptophan prototrophy using the modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2;
  • TRPl disrupting DNA fragment was isolated from ⁇ DB3078 by digestion with NotVPstl. Transformants were selected on BMMD-agar plates and were subsequently patched out on BMMD-agar plates.
  • the genomic PDIl locus amplified by PCR of with primers DS236 and DS303 was achieved as follows: l ⁇ L template genomic DNA, 5 ⁇ L lOXBuffer (Fast Start Taq+Mg, (Roche)), l ⁇ L 1OmM dNTP's, 5 ⁇ L each primer (2 ⁇ M), 0.4 ⁇ L Fast Start Taq, made up to 50 ⁇ L with H 2 O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9700.
  • the Notl expression cassette from the pSAC35-based expression vector, pAYE316 (Sleep et al, 1991, Biotechnology (N Y), 9, 183-187), designed for the secretion of recombinant human albumin, was cloned into the unique NotI-site of the E. coli cloning vector ⁇ BST(+) (Sleep et al, 2001, Yeast, 18, 403-421) to produce plasmid pQC262e.
  • pQC262e was subsequently modified by site-directed mutagenesis (Kunkel et al, 1987, Methods Enzymol, 154, 367-382) with oligonucleotide LRE49 (Table 14) to introduce a unique Asp718I-site immediately upstream of the NotI-site at the ADH1 terminator region of the expression cassette.
  • This produced plasmid pAYE560 ( Figure 21).
  • the pDB2952 PDIl fragment was cloned into the unique Asp718I-site of pAYE560, following digestion with Asp718I, filling the 3 '-recessed ends using T4 DNA polymerase, and calf intestinal alkaline phosphatase treatment of the blunt-ended product.
  • pDB3175 and pDB3176 were produced by cloning the ⁇ 5.1- kb rHA ⁇ PZ>/7 ⁇ Noll expression cassette from pDB3171 into pSAC35. which had been digested with Notl and calf intestinal alkaline phosphatase.
  • pDB3177 and pDB3178 were produced similarly by cloning the ⁇ 5.1- kb Noll rHA— > ⁇ — PDIl expression cassette from pDB3172 into pSAC35 digested with Noll and calf intestinal alkaline phosphatase.
  • Noll expression cassettes for co-expression of recombinant unglycosylated human transferrin (N413Q, N611Q) and the S cerevisiae SKQ2n PDIl gene with the long ( ⁇ 210-bp) promoter the ⁇ 6.1-kb Afl ⁇ l-Sphl fragment from pDB3171 was ligated with the ⁇ 2.4-kb Aflll-Sphl fragment from pDB2928 ( Figure 11 of WO 2005/061718, the contents of which are incorporated herein by reference), thus replacing the rHA coding and adjacent sequences with those for transferrin (N413Q, N61 1 Q) secretion.
  • pDB3179 and pDB3180 were produced by cloning the ⁇ 5.2- kb rTf ⁇ PDIl ⁇ Notl expression cassette from pDB3173 into pSAC35, which had been digested with Notl and calf intestinal alkaline phosphatase.
  • pDB3181 and pDB3182 were produced by cloning the ⁇ 5.2-kb Notl rTf ⁇ — PDIl expression cassette from pDB3174 into pSAC35 digested with Notl and calf intestinal alkaline phosphatase.
  • the yeast strains DXYl (Kerry-Williams el a!., 1998, Yeast, 14, 161-169) and DXYl ⁇ trpl (see Example 13 of WO 2005/061718, the contents of which are incorporated herein by reference) were transformed to leucine prototrophy with pSAC35-based plasmids pAYE316, pDB3175, pDB3176, pDB3177, pDB3178, pDB2931, pDB3179, pDB3180, pDB3181 and pDB3182 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I , protocol 2; (Ito et al, 1983, J Bacteriol, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD-agar plates with appropriate supplements, and were subsequently patched out on BMMD-agar plates with appropriate supplements.
  • DXYl ⁇ trpl [pDB3175], DXYl ⁇ trpl [pDB3176], DXYl Atrpl [pDB3177], DXYl Atrpl [pDB3178], DXYl Atrpl [pDB3179], DXYl Atrpl [pDB3180], DXYl Atrpl [pDB3181], and DXYl Atrpl [pDB3182] were transformed to tryptophan prototrophy using the modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2; (Ito el al, 1983, J Bacteriol., 153, 163; Elble, 1992, Biotechniques, 13, 18)) with a 1.41-kb pdil: TRPl disrupting DNA fragment isolated from pDB3078 by digestion with NotllPstl. (see Example 13 of WO 2005/061718). Transformants were selected on BMMD-agar plates and were
  • CF247 binds in the PDIl promoter region upstream of the disruption site and DS236 binds within the TRPl gene.
  • Table 15 Oligonucleotide Sequencing Primers
  • TRPl -disruptants were inoculated into 1 OmL YEPD in 5OmL shake flasks, and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Genomic DNA was prepared (Lee, 1992, Biotechniques, 12, 677) from the biomass for subsequent use as template DNA in diagnostic PCR.
  • PCRs were performed using a DyadTM DNA Engine Peltier thermal cycler (GRI) as follows; denaturation at 95°C for 4 mins, then 25 cycles of 95°C for 30s, 55°C for 30s and 72°C for 1 min, followed by extension at 72°C for 10 mins.
  • GRI DyadTM DNA Engine Peltier thermal cycler
  • a PCR product of ⁇ 810-bp was only amplified from DNA containing the TRPl gene integrated at the pdil locus.
  • DXYl Atrpl pdil::TRPl containing pDB3175-pDB3182 were successfully identified. As expected, no PCR product was generated for the controls DXYl Atrpl or DXYl Atrpl containing pDB3175-pDB3182.
  • DXYl containing pDB3175-pDB3182 were compared with DXYl Atrpl pdil::TRPl containing pDB3175-pDB3182 for plasmid stability and secretion of recombinant human albumin or transferrin (N413Q. N61 IQ) in YEPD shake flask culture.
  • 1OmL YEPD shake flasks were inoculated with the above strains and grown for 4-days at 30°C, 200rpm. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 3O°C.
  • Plasmid stability was scored as the percentage of colonies able to grow on both media.
  • the results shown in Tables 16 and 17 demonstrated that all of the plasmids pDB3175- pDB3182 were less than 100% stable in DXYl, regardless of the relative orientations of the PDIl and rTf/rHA genes cloned at the SnaBVNotl-site.
  • 100% plasmid stability was determined in all cases in DXYl Atrpl pdil. :TRPl containing pDB3175-pDB3182 .
  • use of PDIl as the sole selectable marker at the SnaBl/Notl-site of pSAC35-based vectors resulted in 100% plasmid stability in rich media.
  • Table 16 Plasmid stability of pSAC35-based vectors containing a recombinant albumin gene and the S cerevisiae PDIl gene at the SnaBI/Notl-site in the UL- region in strains DXYl and DXYl ⁇ trpl pdilr.
  • TRPl. 1OmL YEPD shake flasks were inoculated with DXYl [pDB3175], DXYl [pDB3176], DXYl [pDB3177], DXYl [pDB3178], DXYl Atrpl pdilr.
  • TRPl [pDB3175], DXYl Atrpl pdil::TRPl [pDB3176], DXYl ⁇ irpl pdilr. TRPl [pDB3177], and DXYl Atrpl pdilr. TRPl [pDB3178] were grown for 4-days at 30°C ; 200 ⁇ m. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 30°C. Plasmid stability was scored as the percentage of colonies able to grow on both media.
  • Table 17 Plasmid stability of pSAC35-based vectors containing a recombinant transferrin gene and the S. cerevisiae PDIl gene at the Sn ⁇ BLWo/I-site in the UL- region in strains DXYl and DXYl ⁇ trpl pdil::TRPl.
  • 1 OmL YEPD shake flasks were inoculated with DXYl [pDB3179], DXYl [pDB3180], DXYl [pDB3181], DXYl [pDB3182] : DXYl Atrpl pdilr.
  • TRPl [pDB3179], DXYl ⁇ trpl pdil::TRPl [pDB3180], DXYl ⁇ trpl pdil::TRPl [pDB3181] : and DXYl Atrpl pdil::TRP] [pDB3182] were grown for 4-days at 3O°C, 200rpm. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 30°C. Plasmid stability was scored as the percentage of colonies able to grow on both media.
  • PDIl expression can be modulated, for example by altering the length of the PDIl promoter (Example T) such that an increase in recombinant protein secretion is observed.
  • the long PDIl promoter was used, which was not the preferred promoter length for optimal rHA secretion in the closely related high rHA producing strain, DS569, where a short promoter resulted in increased rHA secretion.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention provides a method for producing a desired protein (such as a desired heterologous protein) comprising: (a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and (b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.

Description

GENE EXPRESSION TECHNIQUE
FIELD OF THE INVENTION
The present application relates to gene expression techniques.
BACKGROUND OF THE INVENTION
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
The class of proteins known as chaperones has been defined by Hard (1996, Nature, 381, 571-580) as a protein that binds to and stabilises an otherwise unstable conformer of another protein and, by controlled binding and release, facilitates its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation.
BiP (also known as GRP78, Ig heavy chain binding protein and Kar2p in yeast) is an abundant -7OkDa chaperone of the hsp 70 family, resident in the endoplasmic reticulum (ER), which amongst other functions, serves to assist in transport in the secretory system and fold proteins.
Protein disulphide isomerase (PDI) is a chaperone protein, resident in the ER that is involved in the catalysis of disulphide bond formation during the post- translational processing of proteins.
Studies of the secretion of both native and foreign proteins have shown that transit from the ER to the Golgi is the rate-limiting step. Evidence points to a transient association of the BiP with normal proteins and a more stable interaction with mutant or misfolded forms of a protein. As a result, BiP may play a dual role in solubilising folding precursors and preventing the transport of unfolded and unassembled proteins. Robinson and Wittrup, 1995, Biotechnol. Prog. 11. 171- 177. have examined the effect of foreign protein secretion on BiP (Kar2p) and PDI protein levels in Saccharomyces cerevisiae and found that prolonged constitutive expression of foreign secreted proteins reduces soluble BiP and PDI to levels undetectable by Western analysis. The lowering of ER chaperone and foldase levels as a consequence of heterologous protein secretion has important implications for attempts to improve yeast expression/secretion systems.
Expression of chaperones is regulated by a number of mechanisms, including the unfolded protein response (UPR).
Using recombinant techniques, multiple PDI gene copies have been shown to increase PDI protein levels in a host cell (Farquhar et al, 1991, Gene, 108. 81-89).
Co-expression of the gene encoding PDI and a gene encoding a heterologous disulphide-bonded protein was first suggested in WO 93/25676, published on 23 December 1993, as a means of increasing the production of the heterologous protein. WO 93/25676 reports that the recombinant expression of antistasin and tick anticoagulant protein can be increased by co-expression with PDI.
This strategy has been exploited to increase the recombinant expression of other types of protein.
Robinson et al, 1994, Bio/Technolog}', 12, 381-384 reported that a recombinant additional PDI gene copy in Saccharomyces cerevisiae could be used to increase the recombinant expression of human platelet derived growth factor (PDGF) B homodimer by ten-fold and Schizosacharomyces pombe acid phosphatase by fourfold. Hayano et al, 1995, FEBS Letters, 377, 505-511 described the co-expression of human lysozyme and PDI in yeast. Increases of around 30-60% in functional lysozyme production and secretion were observed.
Shusta et al, 1998, Nature Biotechnology, 16, 773-777 reported that the recombinant expression of single-chain antibody fragments (scFv) in Saccharomyces cerevisiae could be increased by between 2-8 fold by over- expressing PDI in the host cell.
Bao & Fukuhara, 2001, Gene, 272, 103-110 reported that the expression and secretion of recombinant human serum albumin (rHSA) in the yeast Kluyveromyces lactis could be increased by 15-fold or more by co-expression with an additional recombinant copy of the yeast PDI gene (KlPDIl).
In order to produce co-transformed yeast comprising both a PDI gene and a gene for a heterologous protein, WO 93/25676 taught that the two genes could be chromosomally integrated; one could be chromosomally integrated and one present on a plasmid; each gene could be introduced on a different plasmid; or both genes could be introduced on the same plasmid. WO 93/25676 exemplified expression of antistasin from the plasmid pKH4α2 in yeast strains having a chromosomally integrated additional copy of a PDI gene (Examples 16 and 17); expression of antistasin from the vector K991 with an additional PDI gene copy being present on a multicopy yeast shuttle vector named YEp24 (Botstein et al, 1979, Gene, 8, 17-24) (Example 20); and expression of both the antistasin and the PDI genes from the yeast shuttle vector pCl/1 (Rosenberg et al, 1984. Nature, 312, 77-80) under control of the GALlO and GALl promoters, respectively. Indeed, Robinson and Wittrup, 1995, op. cit., also used the GALl-GALlO intergenic region to express erythropoietin and concluded that production yeast strains for the secretion of heterologous proteins should be constructed using tightly repressible, inducible promoters, otherwise the negative effects of sustained secretion (i.e. lowered detectable BiP and PDI) would be dominant after the many generations of cell growth required to fill a large-scale fermenter.
Subsequent work in the field has identified chromosomal integration of transgenes as the key to maximising recombinant protein production.
Robinson el al, 1994, op. cit., obtained the observed increases in expression of PDGF and S. pombe acid phosphatase using an additional chromosomally integrated PDI gene copy. Robinson et al reported that attempts to use the multi- copy 2μm expression vector to increase PDI protein levels had had a detrimental effect on heterologous protein secretion.
Hayano et al, 1995, op. cit. described the introduction of genes for human lysozyme and PDI into a yeast host each on a separate linearised integration vector, thereby to bring about chromosomal integration.
Shusta et al, 1998, op. cit., reported that in yeast systems, the choice between integration of a transgene into the host chromosome versus the use of episomal expression vectors can greatly affect secretion and, with reference to Parekh & Wittrup, 1997, Biotechnol. Prog., 13, 117-122, that stable integration of the scFv gene into the host chromosome using a δ integration vector was superior to the use of a 2μm-based expression plasmid. Parekh & Wittrup, op. cit., had previously taught that the expression of bovine pancreatic trypsin inhibitor (BPTI) was increased by an order of magnitude using a δ integration vector rather than a 2μm- based expression plasmid. The 2μm-based expression plasmid was said to be counter-productive for the production of heterologous secreted protein.
Bao & Fukuhara, 2001 , op. cit., reported that "It was first thought that the KlPDIl gene might be directly introduced into the multi-copy vector that carried the rHSA expression cassette. However, such constructs were found to severely affect yeast growth and plasmid stability. This confirmed our previous finding that the KlPDIl gene on a multi-copy vector was detrimental to growth of A', lactis cells (Bao et al, 2000)". Bao et al, 2000, Yeast, 16, 329-341, as referred to in the above-quoted passage of Bao & Fukuhara, reported that the KlPDIl gene had been introduced into K. lactis on a multi-copy plasmid. pKan707, and that the presence of the plasmid caused the strain to grow poorly. Bao et al concluded that over-expression of the KlPDIl gene was toxic to K. lactis cells. In the light of the earlier findings in Bao et al, Bao & Fukuhara chose to introduce a single duplication of KlPDIl on the host chromosome.
Against this background, we had previously surprisingly demonstrated that, contrary to the suggestions in the prior art, when the genes for a chaperone protein and a heterologous protein are co-expressed on a 2μm-family multi-copy plasmid in yeast, the production of the heterologous protein is substantially increased.
Our earlier application, which has been published as WO 2005/061718, from which this application claims priority, disclosed a method for producing heterologous protein comprising:
(a) providing a host cell comprising a 2μm-family plasmid, the plasmid comprising a gene encoding a protein comprising the sequence of a chaperone protein and a gene encoding a heterologous protein;
(b) culturing the host cell in a culture medium under conditions that allow the expression of the gene encoding the chaperone protein and the gene encoding a heterologous protein;
(c) purifying the thus expressed heterologous protein from the culture medium; and (d) optionally, lyophilising the thus purified protein.
As discussed above, Bao ei al, 2000. Yeast, 16. 329-341 reported that over- expression of the K. lactis PDI gene KlPDIl was toxic to K. lactis cells. Against this background we have surprisingly found that, not only is it possible to over- express PDI and other chaperones without the detrimental effects reported in Bao el al, but that two different chaperones can be recombinantly over-expressed in the same cell and, rather than being toxic, can increase the expression of proteins, including heterologous proteins, to levels higher than the levels obtained by individual expression of either of the chaperones. This was not expected. On the contrary, in light of the teaching of Bao et al, one would think that over- expression of two chaperones would be even more toxic than the over-expression of one. Moreover, in light of some initial findings which are also reported below in the present application, it was expected that the increases in heterologous protein expression obtained by co-expression with a single chaperone would be at the maximum level possible for the cell system used. Therefore, it was particularly surprising to find that yet further increases in protein expression could be obtained by co-expression of two different chaperones with the protein.
SUMMARY OF THE INVENTION
Accordingly, as a first aspect of the present invention there is provided a method for producing a desired protein, such as a heterologous protein, comprising providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene (optionally the third gene being recombinant) encoding the desired protein (optionally a heterologous protein), wherein the first and second chaperones are different; and culturing the host cell in a culture medium under conditions that allow the expression of the first, second and third genes. Optionally the thus expressed desired protein may. or may not, be purified from the cultured host cell or the culture medium.
Optionally, the thus purified desired protein may. or may not. be lyophilised.
The method may, or may not. further comprise the step of formulating the purified desired protein with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form, in the manner discussed above.
The term '"recombinant gene'" includes nucleic acid sequences that operate independently as "stand alone'" expressible sequences to produce an encoded protein or, in the alternative, nucleic acid sequences introduced that operate in combination with endogenous sequences (such as by integration into an endogenous sequence so as to produce a nucleic acid sequence that is different to the endogenous sequence) within the host to cause increased expression of a target protein.
A ''recombinant gene" is typically a gene that is not naturally found in the context used. For example, a gene that is integrated, at an integration site, into the chromosome of a host organism can be said to be a ''recombinant gene" if it comprises a sequence that does not naturally occur at the integration site. Thus, the "recombinant gene" may, or may not, comprise a non-natural sequence in the coding, regulatory or any other region of the gene, or may, or may not, comprise the sequence of a naturally occurring gene but be introduced into the chromosome of a host organism at an integration site at which that sequence does not naturally occur. The same issues apply, mutatis mutandis, to the insertion of a "recombinant gene" into a plasmid.
The terms "'chromosomaily integrated" and "integrated into the chromosome of the host cell" are well recognised terms of the art. For avoidance of doubt, these terms include the integration of polynucleotide sequences in any inheritable nuclear material that naturally occurs in a host cell, other than for naturally occurring plasmids. Thus, a polynucleotide sequence that is "integrated into the chromosome of the host cell" may. or may not. be integrated into the chromosome of a procaryotic (such as a bacterial) cell, or into any part of the genome of a eucaryotic cell, such as into nuclear genetic material including the chromosome (or, one of the chromosomes), the mitochondrial genome or the chloroplast genome.
The first and second chaperones may, or may not. each individually, be one of the specifically listed chaperones as discussed below, and are a combination of chaperones that, when co-expressed in the same host cell, provide at least an additive effect to the increase in expression of the desired protein. By "additive effect" we mean that the level of expression of the desired protein in the host cell is higher when the first and second recombinant genes are simultaneously co- expressed with the third gene as compared to the same system wherein (i) the first recombinant gene is co-expressed with the third gene in the absence of the expression of the second recombinant gene and (ii) the second recombinant gene is co-expressed with the third gene in the absence of the expression of the first recombinant gene.
The term "chaperone" as used herein refers to a protein that binds to and stabilises an otherwise unstable conformer of another protein, and by controlled binding and release, facilitates its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Accordingly a chaperone is also a protein that is involved in protein folding, or which has chaperone activity or is involved in the unfolded protein response.- Chaperone proteins of this type are known in the art, for example in the Stanford Genome Database (SGD), http://db.yeastgenome.org or http://www.yeastgenome.org. Preferred chaperones are eucaryotic chaperones, especially preferred chaperones are yeast chaperones, including AHAl, CCT2,
CCT3, CCT4, CCT5, CCT6, CCTl, CCT8, CNSl, CPR3, CPR6, EROl, EUGl, FMOL HCHl, HSPlO, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP 82, JEMl, MDJl, MDJ2, MPDl, MP D2, PDIl, PFDl, ABCL APJl, ATPIl, ATP 12, BTTl, CDCSl, CPRl, HSC82, KAR2, LHSl, MGEL MRSlL NOBL ECMlO, SSAl, SSA2, SSA3, SSA4, SSCl, SSE2, SILl, SLSL ORAdI, ORM2, PERl, PTC2, PSEl, UB14 and HACl or a truncated intronless HACl (Valkonen et al. 2003, Applied Environ Micro., 69, 2065), as well as TIM9, PAM18 (also known as TIM14) and TCPl (also known as CCTl)
A chaperone useful in the practice of the present invention may. or may not, be:
• a heat shock protein, such as a protein that is a member of the hsp70 family of proteins (including Kar2p, SSA and SSB proteins, for example proteins encoded by SSAL SSA2, SSA3, SSA4, SSBl and SSB2), a protein that is a member of the HSP90-family, or a protein that is a member of the HSP40-family or proteins involved in their modulation (e.g. SiI Ip). including DNA-J and DNA-J-like proteins (e.g. Jemlp, Mdj2p);
• a protein that is a member of the karyopherin/importin family of proteins, such as the alpha or beta families of karyopherin/importin proteins, for example the karyopherin beta protein PSEl ;
• a protein that is a member of the ORMDL family described by Hjelmqvist et al, 2002, Genome Biology, 3(6), research0027.1-0027.16, such as 0rm2p.
• a protein that is naturally located in the endoplasmic reticulum or elsewhere in the secretory pathway, such as the golgi. For example, a protein that naturally acts in the lumen of the endoplasmic reticulum (ER), particularly in secretory cells, such as PDl • a protein that is transmembrane protein anchored in the ER, such as a member of the ORMDL family described by Hjelmqvist et al, 2002. supra. (for example, Orni2p);
• a protein that acts in the cytosol, such as the hsp70 proteins, including SSA and SSB proteins, for example protein production SSAJ, SSA2, SSA3, SSA4, SSBl and SSB2;
• a protein that acts in the nucleus, the nuclear envelope and/or the cytoplasm, such as Pselp;
• a protein that is "essential'" to the viability of the cell, such as PDI, or a protein encoded by one of the following genes: CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNSl, EROl, HSPlO, HSP 60, PDIl, CDC37, K4R2, MGEl, MRSIl, NOBl, SSCl, TIM9, PAMl 8 and TCPl, or a protein that is an essential karyopherin protein, such as Pselp;
• a protein that is involved in sulphydryl oxidation or disulphide bond formation, breakage or isomerization, or a protein that catalyses thiol:disulphide interchange reactions in proteins, particularly during the biosynthesis of secretory and cell surface proteins, such as protein disulphide isomerases (e.g. Pdilp, Mpdlp), homologues (e.g. Euglp) and/or related proteins (e.g. Mpd2p, Fmolp, Erolp);
• a protein that is involved in protein synthesis, assembly or folding, such as PDI and Ssalp;
• a protein that binds preferentially or exclusively to unfolded, rather than mature protein, such as the hsp70 proteins, including SSA and SSB proteins, for example proteins encoded by SSAl, SSA2, SSA3, SSA4. SSBl and SSB 2;
• a protein that prevents aggregation of precursor proteins in the cytosol, such as the hsp70 proteins, including SSA and SSB proteins, for example proteins encoded by SSAl, SSA2, SSA3, SSA4, SSBl and SSB2:
• a protein that binds to and stabilises damaged proteins, for example Ssalp;
» a protein that is involved in the unfolded protein response or provides for increased resistance to agents (such as tunicamycin and dithiothreitol) that induce the unfolded protein response, such as a member of the ORMDL family described by Hjelmqvist et al, 2002, supra (for example. 0rm2p) or proteins involved in the response to stress (e.g. Ubi4p);
• a protein that is a co-chaperone and/or a protein indirectly involved in protein folding and/or the unfolded protein response (e.g. hsplO4p, Mdj lp);
• a protein that is involved in the nucleocytoplasmic transport of macromolecules, such as Pselp;
• a protein that mediates the transport of macromolecules across the nuclear membrane by recognising nuclear location sequences and nuclear export sequences and interacting with the nuclear pore complex, such as PSEl ;
• a protein that is able to reactivate ribonuclease activity against RNA of scrambled ribonuclease as described in as described in EP 0 746 611 and Hillson el al, 1984, Methods En∑ymol, 107, 281-292, such as PDI; β a protein that has an acidic pi (for example. 4.0-4.5). such as PDI;
• a protein that is a member of the Hsp70 family, and optionally possesses an N-terminal ATP-binding domain and a C-terminal peptide-binding domain, such as S sal p.
• a protein that is a peptidyl-prolyl cis-trans isomerases (e.g. Cpr3p, Cprόp);
• a protein that is a homologue of known chaperones (e.g. Hspl Op);
• a protein that is a mitochondrial chaperone (e.g Cpr3p);
• a protein that is a cytoplasmic or nuclear chaperone (e.g Cnslp);
• a protein that is a membrane-bound chaperone (e.g. 0rm2p, Fmolp);
• a protein that has chaperone activator activity or chaperone regulatory activity (e.g. Ahalp, Haclp, Hchlp);
• a protein that transiently binds to polypeptides in their immature form to cause proper folding transportation and/or secretion, including proteins required for efficient translocation into the endoplasmic reticulum (e.g. Lhslp) or their site of action within the cell (e.g. Pselp);
• a protein that is a involved in protein complex assembly and/or ribosome assembly (e.g. Atpl lp, Pselp, Noblp);
• a protein of the chaperonin T-complex (e.g. Cct2p);
• a protein of the prefoldin complex (e.g. Pfdlp); • a mitochondrial intermembrane space protein such as Tim9p;
• a protein that can form a complex., in vivo, with Mrsl lp/TimlOp. such as Tim9p;
• a protein that is involved in the mediation of import and insertion of polytopic inner membrane proteins, such as Tim9p;
• a protein that can prevent the aggregation of incoming proteins, such as Tim9p;
• a protein that can be a functional constituent of the mitochondrial import motor associated with presequence translocase (along with Ssclp, Tim44p, Mgelp and Pamlόp) such as Pamlδp;
• a protein that can stimulate the ATPase activity of Ssclp, such as to drive mitochondrial import, such as PamlSp;
• a protein that contains a J domain, such as Pam 18p;
• a protein that can act as an alpha subunit of chaperonin-containing T- complex, which mediates protein folding in the cytosol, such as Tcplp;
• a protein that can play a role in the in maintenance of an actin cytoskeleton, such as Tcplp; or
• a protein that is, or is a homolog to, a Drosophila melanogasier or mouse tailless complex polypeptide, such as Tcplp. A preferred chaperone is protein disulphide isomerase (PDI) or a fragment or variant thereof having an equivalent ability to catalyse the formation of disulphide bonds within the lumen of the endoplasmic reticulum (ER). By "PDF"' we include any protein having the ability to reactivate the ribonuclease activity against RNA of scrambled ribonuclease as described in EP 0 746 61 1 and Hillson el al, 1984, Methods En∑ymol, 107, 281-292.
PDl is an enzyme which typically catalyzes thiokdisulphide interchange reactions, and is a major resident protein component of the ER lumen in secretory cells. A body of evidence suggests that it plays a role in secretory protein biosynthesis (Freedman, 1984, Trends Biochem. Sci., 9, 438-41) and this is supported by direct cross-linking studies in situ (Roth and Pierce, 1987, Biochemistry, 26, 4179-82). The finding that microsomal membranes deficient in PDI show a specific defect in cotranslational protein disulphide (Bulleid and Freedman. 1988. Nature, 335, 649- 51) implies that the enzyme functions as a catalyst of native disulphide bond formation during the biosynthesis of secretory and cell surface proteins. This role is consistent with what is known of the enzyme's catalytic properties in vitro: it catalyzes thiol: disulphide interchange reactions leading to net protein disulphide formation, breakage or isomerization, and can typically catalyze protein folding and the formation of native disulphide bonds in a wide variety of reduced, unfolded protein substrates (Freedman et ah, 1989, Biochem. Soc. Symp., 55, 167- 192). PDI also functions as a chaperone since mutant PDI lacking isomerase activity accelerates protein folding (Hayano et al, 1995, FEBS Letters, 377, 505- 51 1). Recently, sulphydryl oxidation, not disulphide isomerisation was reported to be the principal function of Protein Disulphide Isomerase in 5. cerevisiae (Solovyov et al, 2004, J. Biol. Chem, 279 (33) 34095-34100). The DNA and amino acid sequence of the enzyme is known for several species (Scherens et al, 1991, Yeast, 7, 185-193; Farquhar et al, 1991, Gene, 108, 81-89; EP074661 ; EP0293793; EP0509841) and there is increasing information on the mechanism of action of the enzyme purified to homogeneity from mammalian liver (Creighton et al, 1980, J. MoI. Biol, 142, 43-62; Freedman et al, 1988, Biochem. Soc. Trans., 16, 96-9; Gilbert, 1989, Biochemistry, 28; 7298-7305; Lundstrom and Holmgren, 1990, J. Biol. Chem , 265; 9114-9120: Hawkins and Freedman., 1990, Biochem. J., 275, 335-339). Of the many protein factors currently implicated as mediators of protein folding, assembly and translocation in the cell (Rothman. 1989. Cell. 59, 591-601), PDI has a well-defined catalytic activity.
The deletion or inactivation of the endogenous PDI gene in a host results in the production of an inviable host. In other words, the endogenous PDI gene is an '"essential"' gene.
PDI is readily isolated from mammalian tissues and the homogeneous enzyme is a homodimer (2x57 kD) with characteristically acidic pi (4.0-4.5) (Hillson el al, 1984, op. cit.). The enzyme has also been purified from wheat and from the alga Chlamydomonas reinhardii (Kaska et al, 1990. Biochem. J, 268, 63-68), rat (Edman et al, 1985, Nature, 317, 267-270), bovine (Yamauchi et al, 1987, Biochem. Biophys. Res. Comm., 146, 1485-1492). human (Pihlajaniemi et al, 1987, EMBO J, 6, 643-9), yeast (Scherens el al, supra; Farquhar et al, op. cit.) and chick (Parkkonen et al, 1988, Biochem. J., 256, 1005-1011). The proteins from these vertebrate species show a high degree of sequence conservation throughout and all show several overall features first noted in the rat PDI sequence (Edman et ah, 1985, op. cit.).
Preferred PDI sequences include those from humans and those from yeast species, such as 5. cerevisiae.
A yeast protein disulphide isomerase precursor, PDIl, can be found as Genbank accession no. CAA42373 or BAA00723 and has a sequence of 522 amino acids as described in WO 2005/061718, the contents of which are incorporated herein by reference. An alternative yeast protein disulphide isomerase sequence can be found as Genbank accession no. CAA38402, which has a sequence of 530 amino acids as described in WO 2005/061718. the contents of which are incorporated herein by reference.
The alignment of these sequences (the sequence of Genbank accession no. CAA42373 or BAA00723 first, the sequence of Genbank accession no. CAA38402 second) in WO 2005/061718, the contents of which are incorporated herein by reference, shows that the differences between these two sequences are a single amino acid difference at position 114 (highlighted in bold) and that the sequence defined by Genbank accession no. CAA38402 contains the additional amino acids EADAEAEA at positions 506-513.
Variants and fragments of the above PDI sequences, and variants of other naturally occurring PDI sequences are also included in the present invention. A "variant", in the context of PDI, refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may, or may not, still be different but would not be unobvious over the ones of the original protein.
By "conservative substitutions" is intended combinations such as VaI, He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Tip. Preferred conservative substitutions include GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
A "variant" typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the polypeptide from which it is derived.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
A '"fragment", in the context of PDI, refers to a protein wherein at one or more positions there have been deletions. Thus the fragment may. or may not. comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%, more preferably up to 90%, even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature PDI protein. Particularly preferred fragments of PDI protein comprise one or more whole domains of the desired protein.
A fragment or variant of PDl may, or may not, be a protein that, when expressed recombinant!)' in a host cell, can complement the deletion of the endogenously encoded PDI gene in the host cell, such as S. cerevisiae, and may, or may not, for example, be a naturally occurring homolog of PDI, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
Where the first chaperone is PDI, particularly mammalian or yeast PDI, then in one embodiment the second chaperone is not an hsp70 chaperone protein (such as yeast KAR2, HSP70, BiP, SSA1-4, SSBl , SSCl, SSDl or a eucaryotic hsp70 protein such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP), glucose-regulated proteins 75, 78 and 80
(GPR75, GRP78 and GRP80) and the like). Specifically in one embodiment the first chaperone is not yeast PDI when the second chaperone is yeast KAR2. Specifically in another embodiment the first chaperone is not mammalian PDI when the second chaperone is mammalian BiP.
Alternatively, where the first and second chaperones are, for example. PDI, particularly mammalian or yeast PDl, and an hsp70 chaperone protein as described above, respectively, then the desired protein may be a heterologous protein that may or may not be a protein selected from -
• mammalian gene products such as enzymes, cytokines, growth factors, hormones, vaccines, antibodies and the like; erythropoietin, insulin, somatotropin, growth hormone releasing factor, platelet derived growth factor, epidermal growth factor, transforming growth factor α, transforming growth factor β, epidermal growth factor, fibroblast growth factor, nerve growth factor, insulin-like growth factor I, insulin-like growth factor II, clotting Factor VIII, superoxide dismutase, α-interferon, γ-interferon, interleukin-1, interleukin-2, interleukin-3, interleukin-4. interleukin-5, interleukin-6, granulocyte colony stimulating factor, multi- lineage colony stimulating activity, granulocyte-macrophage stimulating factor, macrophage colony stimulating factor, T cell growth factor, lymphotoxin and the like; or human gene products; • any gene product which can be used as a vaccine, including any structural, membrane-associated, membrane-bound or secreted gene product of a mammalian pathogen, including viruses, bacteria, single-celled or multi- celled parasites which can infect or attack a mammal, in particular viruses such as human immunodeficiency virus (HIV), R. rickettsii, vaccinia, Shigella, poliovirus, adenovirus, influenza, hepatitis A, hepatitis B, dengue virus, Japanese B encephalitis, Varicella zostei\ cytomegalovirus, hepatitis A, rotavirus, as well as vaccines against viral diseases like Lyme disease, . measles, yellow fever, mumps, rabies, herpes, influenza, parainfluenza and the like; or bacteria such as Vibrio cholerae, Salmonella typhi, Bordetella pertussis, Streptococcus pneumoniae, Hemophilus influenza, Clostridium tetcmi. Corynebacferium diphtheriae, Mycobacterium leprae, Ηeisseriaqonorrhoeae, Neisseiϊameningitidis, Coccidioides immitis and the like.
Another preferred chaperone is a protein comprising the sequence of a protein encoded by the gene SSAl, or a fragment or variant thereof having an equivalent chaperone-like activity. SSAl . also known as YGlOO, is located on chromosome I of the S. cerevisiae genome and is 1.93-kbp in size.
One published protein sequence of the protein encoded by the gene SSAl is as described in WO 2005/061718, the contents of which are incorporated herein by reference.
A published coding sequence for SSAl is also described in WO 2005/061718. the contents of which are incorporated herein by reference, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical protein product.
The protein Ssalp belongs to the Hsp70 family of proteins and is resident in the cytosol. Hsp70s possess the ability to perform a number of chaperone activities; aiding protein synthesis, assembly and folding; mediating translocation of polypeptides to various intracellular locations, and resolution of protein aggregates (Becker & Craig, 1994, Eur. J. Biochem. 219, 11-23). Hsp70 genes are highly conserved, possessing an N-terminal ATP -binding domain and a C- terminal peptide-binding domain. Hsp70 proteins interact with the peptide backbone of, mainly unfolded, proteins. The binding and release of peptides by hsp70 proteins is an ATP-dependent process and accompanied by a conformational change in the hsp70 (Becker & Craig, 1994, supra).
Cytosolic hsp70 proteins are particularly involved in the synthesis, folding and secretion of proteins (Becker & Craig, 1994, supra). In S. cerevisiae cytosolic hsp70 proteins have been divided into two groups; SSA (SSA 1-4) and SSB (SSB
1 and 2) proteins, which are functionally distinct from each other. The SSA family is '"essential'" in that at least one protein from the group must be active to maintain cell viability (Becker & Craig. 1994, supra). Cytosolic hsp70 proteins bind preferentially to unfolded and not mature proteins. This suggests that they
' prevent the aggregation of precursor proteins, by maintaining them in an unfolded state prior to being assembled into multimolecular complexes in the cytosol and/or facilitating their translocation to various organelles (Becker & Craig, 1994, supra). SSA proteins are particularly involved in posttranslational biogenesis and maintenance of precursors for translocation into the endoplasmic reticulum and mitochondria (Kim el ai, 1998, Proc. Natl. Acad. ScI USA. 95, 12860-12865; Ngosuwan et al., 2003, J. Biol. Chem. 278 (9), 7034-7042). Ssalp has been shown to bind damaged proteins, stabilising them in a partially unfolded form and allowing refolding or degradation to occur (Becker & Craig, 1994, supra; Glover & Lindquist 1998, Cell. 94, 73-82).
Demolder et al, 1994. J. Biotechnol., 32, 179-189 reported that over-expression of SSAl in yeast provided for increases in the expression of a recombinant chromosomally integrated gene encoding human interferon-β. There is no suggestion that increases in heterologous gene expression could be achieved if SSAl and human interferon-β were to be encoded by recombinant genes on the same plasmid. In fact, in light of more recent developments in the field of over- expression of chaperones in yeast (e.g. Robinson et al, 1994, op. cit.; Hayano et al, 1995, op. cit., Shusta et al, 1998, op. cit; Parekh & Wittrup, 1997, op. cit.; Bao & Fukuhara, 2001, op. cit.; and Bao et al, 2000, op. cit ) the skilled person would have been disinclined to express SSAl from a 2μm-family plasmid at all, much less to express both SSAl and a heterologous protein from a 2μm-family plasmid in order to increase the expression levels of a heterologous protein. Variants and fragments of is a protein comprising the sequence of a protein encoded by the gene SSAJ are also included in the present invention. A "variant", in the context of a protein encoded by the gene SSAJ, refers to a protein having the sequence of native Ssalp other than at one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed. "'Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
By "conservative substitutions" is intended combinations such as VaI. He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Trp. Preferred conservative substitutions include GIy, Ala; VaI. He. Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg: and Phe, Tyr.
A "variant" of Ssalp typically has at least 25%. at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Ssalp.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
A '"fragment", in the context of Ssalp, refers to a protein having the sequence of native Ssalp other than for at one or more positions where there have been deletions. Thus the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%. more preferably up to 90%, even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature Ssalp protein. Particularly preferred fragments of SSAl protein comprise one or more whole domains of the desired protein.
A fragment or variant of Ssalp may. or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenously encoded SSAl gene (or homolog thereof) in the host cell and may, or may not. for example, be a naturally occurring homolog of Ssalp, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
Another preferred chaperone is protein comprising the sequence of a protein encoded by the PSEJ gene, or a fragment or variant thereof having equivalent chaperone-like activity.
PSEl, also known as KAPl 2], is an essential gene, located on chromosome XIII.
A published protein sequence for the protein Pselp is as described in WO 2005/061718, the contents of which are incorporated herein by reference.
A published nucleotide coding sequence of PSEl is also described in WO 2005/061718, the contents of which are incorporated herein by reference, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical protein product.
The PSEl gene is 3.25-kbp in size. Pselp is involved in the nucleocytoplasmic transport of macromolecuies (Seedorf & Silver, 1997, Proc. Natl. Acad. Sci. USA. 94, 8590-8595). This process occurs via the nuclear pore complex (NPC) embedded in the nuclear envelope and made up of nucleoporins (Ryan & Wente, 2000. Curr. Opin. Cell Biol. 12, 361-371). Proteins possess specific sequences that contain the information required for nuclear import, nuclear localisation sequence (NLS) and export, nuclear export sequence (NES) (Pemberton et al., 1998, Curr. Opin. Cell Biol. 10, 392-399). Pselp is a karyopherin/importin, a group of proteins, which have been divided up into α and β families. Karyopherins are soluble transport factors that mediate the transport of macromolecules across the nuclear membrane by recognising NLS and NES, and interact with and the NPC (Seedorf & Silver, 1997, supra; Pemberton el al, 1998, supra; Ryan & Wente, 2000, supra). Translocation through the nuclear pore is driven by GTP hydrolysis, catalysed by the small GTP -binding protein, Ran (Seedorf & Silver, 1997, supra). Pselp has been identified as a karyopherin β. 14 karyopherin β proteins have been identified in S. cerevisiae, of which only 4 are '"essential". This is perhaps because multiple karyopherins may mediate the transport of a single macromolecule (Isoyama et al, 2001, J. Biol. Chem. 276 (24), 21863-21869). Pselp is localised to the nucleus, at the nuclear envelope, and to a certain extent to the cytoplasm. This suggests the protein moves in and out of the nucleus as part of its transport function (Seedorf & Silver, 1997, supra). Pselp is involved in the nuclear import of transcription factors (Isoyama el al.,
2001, supra; Ueta et al, 2003, J. Biol. Chem. 278 (50), 50120-50127), histones (Mosammaparast et al., 2002, J. Biol. Chem. 277 (1), 862-868), and ribosomal proteins prior to their assembly into ribosomes (Pemberton et al., 1998, supra). It also mediates the export of mRNA from the nucleus. Karyopherins recognise and bind distinct NES found on RNA-binding proteins, which coat the RNA before it is exported from the nucleus (Seedorf & Silver, 1997, Pemberton et al., 1998, supra).
As nucleocytoplasmic transport of macromolecules is essential for proper progression through the cell cycle, nuclear transport factors, such as Pselp are novel candidate targets for growth control (Seedorf & Silver, 1997, supra). Overexpression of Pselp (protein secretion enhancer) in S cerevisiae has also been shown to increase endogenous protein secretion levels of a repertoire of biologically active proteins (Chow el al., 1992; J. Cell. ScI 101 (3), 709-719). There is no suggestion that increases in heterologous gene expression could be achieved if Pselp and a heterologous protein were both to be encoded by recombinant genes on the same plasmid. In fact, in light of more recent developments in the over-expression of chaperones in yeast (e.g. Robinson et al, 1994, op. cit. ; Hayano et al, 1995, op. cit.; Shusta et al, 1998, op. cit; Parekh & Wittrup, 1997, op. cit ; Bao & Fukuhara, 2001, op. cit; and Bao el al, 2000, op. cit ) the skilled person would not have attempted to over-express a PSEl gene from a 2μm-family plasmid at all, much less to express both Pselp and a heterologous protein from a 2μm-family plasmid in order to increase the expression levels of a heterologous protein.
Variants and fragments of Pselp are also included in the present invention. A "variant'", in the context of Pselp, refers to a protein having the sequence of native Pselp other than for at one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
By "conservative substitutions" is intended combinations such as VaI, He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe, Tyr, Tip. Preferred conservative substitutions include GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. A '"variant" of Pselp typically has at least 25%. at least 50%. at least 60% or at least 70%. preferably at least 80%. more preferably at least 90%. even more preferably at least 95%. yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Pselp.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
A '"fragment", in the context of Pselp, refers to a protein having the sequence of native Pselp other than for at one or more positions where there have been deletions. Thus the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or 50%, typically up to 60%, more typically up to 70%, preferably up to 80%, more preferably up to 90%. even more preferably up to 95%, yet more preferably up to 99% of the complete sequence of the full mature Pselp protein. Particularly preferred fragments of Pselp protein comprise one or more whole domains of the desired protein.
A fragment or variant of Psel p may, or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenous PSEl gene in the host cell and may, or may not, for example, be a naturally occurring homolog of Pselp, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
Another preferred chaperone is a protein comprising the sequence of a protein encoded by the ORA42 gene, or a fragment or variant thereof having equivalent chaperone-like activity. ORM2. also known as YLR350W. is located on chromosome XII (positions 828729 to 829379) of the S. cerevisiae genome and encodes an evolutionarily conserved protein with similarity to the yeast protein Ormlp. Hjelmqλ'ist el al, 2002, Genome Biology, 3(6), research 0027.1-0027.16 reports that 0RM2 belongs to gene family comprising three human genes (ORMDLl, ORMDL2 and ORMDL3) as well as homologs in microsporidia, plants. Drosophila, urochordates and vertebrates. The ORMDL genes are reported to encode transmembrane proteins anchored in the proteins endoplasmic reticulum (ER).
The protein Orm2p is required for resistance to agents that induce the unfolded protein response. Hjelmqvist et al, 2002 (supra) reported that a double knockout of the two S. cerevisiae ORMDL homologs (ORMl and ORMI) leads to a decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol.
One published sequence of Orm2p is as described in WO 2005/061718, the contents of which are incorporated herein by reference.
The above protein is encoded in S. cerevisiae by the coding nucleotide sequence also as described in WO 2005/061718, the contents of which are incorporated herein by reference, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical protein product.
Variants and fragments of Orm2p are also included in the present invention. A "variant", in the context of Orm2p, refers to a protein having the sequence of native
0rm2p other than for at one or more positions where there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
By "conservative substitutions'' is intended combinations such as VaI, He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys. Arg, His; and Phe, Tyr, Trp. Preferred conservative substitutions include GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn. GIn; Ser. Thr; Lys, Arg; and Phe, Tyr.
A "variant" of Orm2p typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the sequence of native Orm2p.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, as discussed below. Such variants may, or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
A "fragment", in the context of Orm2p, refers to a protein having the sequence of native Orm2p other than for at one or more positions where there have been deletions. Thus the fragment may, or may not, comprise at most 5, 10, 20, 30, 40 or
50%, typically up to 60%, more typically up to 70%, preferably up to 80%, more preferably up to 90%. even more preferably up to 95%, yet more preferably up to
99% of the complete sequence of the full mature Orm2p protein. Particularly preferred fragments of Orm2p protein comprise one or more whole domains of the desired protein.
A fragment or variant of 0rm2p may, or may not, be a protein that, when expressed recombinantly in a host cell, such as S. cerevisiae, can complement the deletion of the endogenous 0RM2 gene in the host cell and may, or may not, for example, be a naturally occurring homolog of 0rm2p, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eucaryote such as a human or other vertebrate, or animal or by a plant.
A gene encoding a protein comprising the sequence of a chaperone may, or may not, be formed in a like manner to that discussed below for genes encoding heterologous proteins, with particular emphasis on combinations of ORFs and regulatory regions.
Thus, one preferred chaperone is protein disulphide isomerase; another preferred chaperone is 0rm2p or a fragment or variant thereof. In a particularly preferred embodiment, the first and second chaperones are protein disulphide isomerase and 0rm2p or a fragment or variant thereof.
Further preferred combinations for the first and second chaperones. respectively, may, or may not, be encoded by the genes AHAl and CCT2; AHAl and CCT3; AHAl and CCT4\ AHAl and CCT5; AHAl and CCT6; AHAl and CCT7; AHAl and CCT8; AHAl and CNSl; AHAl and CPR3; AHAl and CPR6; AHAl and EROl: AHAl and EUGl; AHAl and FMOl; AHAl and HCHl; AHAl and HSPlO; AHAl and HSPl 2; AHAl and HSP104; AHAl and HSP26; AHAl and HSP30; AHAl and HSP42; AHAl and HSP60; AHAl and HSP78; AHAl and HSP82; AHAl and JEMl; AHAl and MDJl; AHAl and MDJ2; AHAl and MPDl; AHAl and MPD2; AHAl and PDH; AHAl and PFDl; AHAl and ABCl; AHAl and APJl; AHAl and ATPl 1; AHAl and ATP12; AHAl and BTTl; AHAl and CDC37; AHAl and CPR7; AHAl and HSC82; AHAl and KAR2; AHAl and LHSl; AHAl and MGEl; AHAl and MRSIl; AHAl and NOBl; AHAl and ECMlO; AHAl and SSAl; AHAl and SSA2; AHAl and SSA3; AHAl and SSA4; AHAl and SSCl; AHAl and SSE2; AHAl and SILl; AHAl and SLSV; AHAl and <9i?Mi; Λ/£4V and 0RM2; AHAl and rø?V; Λ/i4i and PTC2; AHAl and />S£i; AHAl and E/Rrø; Af£4V and HACl or a truncated intronless HACl; CCT2 and <XT5; CCT2 and CCT4; CCT2 and CCT5; CCT2 and CCJd; CCT2 and CCT7; CCT2 and CC7S;
CCT2 and CAW: CC77 and CPR 3; CCT2 and CP7?5; CCT2 and £7? 07; CCT2 and EUGl; CCT2 and FMOl; CCT2 and HCHl; CCT2 and HSPlO; CCT2 and HSP 12; CCT2 and HSP 104; CCT2 and HSP26; CCT2 and HSP30; CCT2 and HSP 42; CCT2 and HSP60; CCT2 and #SP7S; CCT2 and HSP82: CCT2 and J£M7; CCT2 and MZ)Ji; CCT2 and MDJ2; CCT2 and MPD7; COT and MPD2; CC72 and PDIl ; CC7L? and PFD7 ; CCT2 and ^5C7 ; CCT2 and ΛPJ7 : CC72 and ATPIl; CCT2 and 47P.72; CCT2 and 57Ti; CC77 and CDC37; CCT2 and CPi? 7; CC72 and HSC82; CCT2 and ΛL4i22; CCT2 and LHSi; CC77 and MGEl; CCT2 and Mi?Sii; CCI2 and NOBl; CCT2 and ECMiO; CCT2 and SSΛi; CC72 and SSA2; CCT2 and S&4J; CC72 and SSA4; CCT2 and SSCi; CCT2 and SS£2; CC72 and SILl ; CCT2 and SLSl ; CCT2 and ORMl ; CCT2 and Oi?M2; CCT2 and P£i?i ; CCI2 and PTC2; CCT2 and PS£i; CCT2 and t/ZW; CC72 and HACl or a truncated intronless HACl; CCT3 and CC7V; CC75 and CCT5; CCT3 and CC7(5; CCT3 and CC77; CC73 and CCT8; CCT3 and CNSi; CC73 and CPR3; CCT3 and CPi? 6; CCT3 and £i?Oi; CCPi and EUGl; CCT3 and FMOi; CCT3 and HCHi; CCπ and HSPlO; CCT3 and HSPi2; CCT3 and HSP 104; CCT3 and tf£P25; CC73 and HSP30; CCΗ and HSP 42; CCT3 and HSP60; CCT3 and HS1P 75; CC75 and HSP82; CCT3 and J£Mi; CC75 and MDJl; CCT3 and Λ4DJ2; CCT3 and MPDi; CC75 and MPD2; CCT3 and PZ)H; CC73 and PFDl; CCT3 and ΛPCi; CCT3 and .4PJi; CC73 and ATPIl; CCT3 and Λ7Pi2; CC73 and BTTl; CCπ and CDC37; CCT3 and CPi? 7; CC73 and HSC82; CCT3 and iζ^i?2; CCT3 and IHSi; CC73 and MGEl; CCT3 and Mi?Sii; CC73 and NOBl; CCT3 and ECMlO; CCT3 and SS^i; CC73 and SSA2; CCT3 and SS^3; CC73 and SSA4; CCT3 and SSCi; CCT3 and SS£2; CC73 and SILl; CCT3 and SISi; CC73 and <9i?Mi; CCT3 and (9i?M2; CCT3 and P£βi; CC73 and PTC 2; CCT3 and PS/ii; CCT3 and C/B74; CC73 and HACl or a truncated intronless HACl; CCT4 and CCT5; CC7¥ and CCT6; CCT4 and CC77; CCT4 and CC7S; CC74 and CNSl; CCT4 and CPi?3; CC7¥ and CPR6; CCT4 and £i?Oi; CCT4 and ££/Gi; CC74 and FMOl; CCT4 and HCHi; CC74 and HSPlO; CCT4 and HSPi 2; CCT4 and HSPl 04; CCT4 and ΛSP25; CCT^ and HSP30; CCT4 and USP 42; CCT4 and HSP60; CCT4 and HSP 75; CCT4 and HSP52; CC7V and JEMl ; CCT4 and MDJi ;
CCT4 and MDJ2; CC7¥ and MPDl; CCT4 and MPD2; CCT4 and PDH: CC7V and PFDl; CCT4 and ABCJ: CCT4 and APJl: CCT4 and ATPl 7; CCT4 and ATP 12: CCT4 and 57T7: CCT4 and CZ)CJ 7; CCW and CPR7; CCT4 and ZZ5CS2; CCW and KAR2; CCT4 and XHSi; CCT4 and MG£7; CCW and MRSIl: CCT4 and 7V057: CCW and ECMlO: CCT4 and 5547; CCT4 and 5&42; CCW and 5545; CCW and 5544; CCT4 and 55C7; CCW and SSE2: CCT4 and 5/17; CCW and SLSl; CCT4 and <9i?Mi; CCT4 and O7?M2; CCW and PERl; CCT4 and P7C2; CCW and PSEl: CCT4 and C7574; CCW and 7i4CZ or a truncated intronless HACl; CCT5 and CCT6: CCT5 and CCZ7; CC-TJ and CCT8; CCT 5 and CN57; CCT5 and CP#3; CC-TJ and CPR6; CCT5 and £7?<9Z; CCH and EUGl; CC7J and FMOl ; CCT5 and HCHl ; CCT5 and Z/5PZ 0; CCT5 and 77SP72; CCrJ and HSP 104; CCT5 and ZZ5P26: CCT5 and 7/SP30; CC-TJ and HSP 42; CCT5 and TfSPtfO; CCrJ and HSP78; CCT5 and Z/5PS2; CCT5 and J£M7; CCrJ and MDJl; CCT5 and MDJ2; CCrj and MPDl; CCT5 and MPD2; CCT5 and P73/7; CC75 and PFDl; CCT5 and ^5Ci; CCrj and APJl; CCT5 and ΛZPZi; CCT5 and y47Pi 2; CCrj and 5TPZ ; CCT5 and CZ)C37; CCIJ and CPR 7; CCT5 and HSC82; CCT 5 and iC4i?2; CCPi and LHSl; CCT5 and MGZf 7; CCT 5 and M7?5Zi; CC-TJ and NOBJ; CCT5 and ECMlO; CCT5 and 55.47; CCTJ and SS42; CCT5 and S&43; CCT5 and SSA4; CCT5 and 55C7; CC7b" and SSE2; CCT5 and 5ZI7; CCT5 and 5157; CCZb" and ORMl; CCT5 and Oi?M2; CC-TJ and P£7?7; CCT5 and PTC2; CCT5 and P5£Z; CCTJ and UB14; CCT5 and Zi4CZ or a truncated intronless Z£4C7; CCT6 and CCZ7; CCP(5 and CCT8; CCT6 and CN5Z; CCZ1^ and CPRS; CCT6 and CP7?(5; CCT6 and £7?<97; CCP6 and EUGl; CCT6 and FMOZ; CCZtf and HCHl; CCT6 and ZZ5P70; CCT6 and ZZ5P72; CCZtf and HSP 104; CCT6 and ZZ5P25; CCPd and HSP30; CCT6 and ZZ5P¥2; CCT6 and 7/5P60; CCP5 and HSP 78; CCT6 and ZZ5PS2; CCT6 and JZiMZ; CCPd and MDJ7; CCT6 and MZ3J2; CCPd and MPDl; CCT6 and MPD2; CCT6 and PDZi; CCZtf and PFDl; CCT6 and ΛZ3CZ; CCPd and APJl; CCT6 and ΛSTP77; CC7tf and ATP12; CCT6 and BTTl; CCT6 and CZ)C37; CCP^ and CP R7; CCT6 and ZZ5C52; CCT6 and iC4i?2; CCTd and LHSl; CCT6 and MG/i7; CC7(Ϊ and MRSIl; CCT6 and NOBl; CCT6 and ECMlO; CCT6 and 5&47; CCI5 and 55,42; CCT6 and 5S43;
CCr^ and 55Λ4; CCT6 and 55C7; CCT5 and 55£2; CCT6 and 5ZL7; CCT6 and SLSJ; CCT6 and ORMl: CCT6 and ORM2; CCT6 and PERl: CCT6 and PTC2: CCT6 and PSEl; CCT 6 and UBI4; CCT6 and HACl or a truncated intronless PL4CT; CC77 and CCT8; CCTl and CTVSi; CCTl and CPTU; CCP7 and CPR6; CCTl and £7?O7; CCP7 and EUGl; CCTl and FM07; CCTl and PO77: CCP7 and HSPlO; CCTl and HSP72; CCTl and HSP 104; CCTl and 7PSP26; CC77 and /75PJ0; CCP7 and HSP 42; CCTl and /7SP<50; CCTl and TP5P7S; CCP7 and HSP82; CCTl and J£M7; CCP7 and MDJ7; CCTl and MDJ2; CCTl and MPD7; CCP7 and MPD2; CCTl and P73/7: CCP7 and PFDl; CCTl and ABCT; CCTl and ΛPJT; CCP7 and ATPIl; CCTl and .4PPT2: CCP7 and 737T7; CCTl and CDCiI; CCTl and CPT? 7; CCT7 and HSC82; CCTl and iC47?2; CCTl and Z7P5T; CCP7 and MGEl; CCTl and AfRSi i; CC77 and 7VO87; CCTl and ECMlO; CCTl and S&47; CCT7 and SSA2; CCTl and 55^3; CC77 and SSA4; CCTl and 55C7; CCTl and 55752; CC77 and SILl; CCTl and 5Z57; CCr7 and ORMl; CCTl and O7WI/2; CCTl and P£7?7; CCT7 and PTC2; CCTl and P5£7; CC77 and UBI4; CCT7 and HACl or a truncated intronless HACl; CCT8 and CNS7; CCPS and CPR3; CCT8 and CP7?5; CCT8 and £7?O7; CCP5 and EUGl; CCT8 and FM07; CCP(S and HCHl; CCT8 and H5P70; CCT8 and HSP72; CCP5 and HSP 104; CCT8 and /PSP26; CCPS and HSP30; CCT8 and 775P^/2; CCT8 and TPSPdO; CCPS and HSP18; CCT8 and 7P5PS2; CCPS and JEMl; CCT8 and ΛfP>J7; CCT8 and MDJ2; CCPS and MPD7; CCT8 and MPD2; CCT8 and PT3/7; CCPS and PPD7; CCT8 and ^5C7; CCPS and APJ7; CCT8 and ^PP77; CCT8 and ΛPP72; CCPS and 5PP7; CCT8 and CZ)Ci 7; CCPS and CPT? 7; CCT8 and H5CS2; CCT8 and TL4T?2; CCPS and IPPS7; CCT8 and 7l/GTi7; CCPS and MT?STT; CCT8 and N0T5T; CCT8 and ECMlO; CCT8 and ££17; CCPS and S&42; CCPS and SSA3; CCPS and SSA4; CCT8 and S5CT; CCPS and SSE2; CCT8 and 5/ZT; CCT8 and 5157; CCPS and ORMl; CCT8 and <9£M2; CCPS and P£T?7; CCT8 and PPC2; CCT8 and P5£7; CCPS and UBI4; CCT8 and /i4C7 or a truncated intronless HΛCT; CN5T and CPT?5; CNSl and CP7?(5; CNSl and £7?<97; C7V57 and EUGl; CNSl and FMOT; C7V5T and HCHl; CNSl and TT5PT0; CTVSi and HSP 12; CNSl and HSP104; CNSl and TP5P25; CTVSi and HSP30; CNSl and HSP42; CNSl and
ΛSP60; C7V57 and 7P5P7S; CTVSi and HSP82; CNSl and JEMl; CNSl and MDJT; CNSl and MDJ2; CNSl and MPDl; CNSl and MPD2: CNSl and PDIl \ CNSl and PFDl: CNSl and ABCl: CNSl and APJl: CNSl and ATPIl: CNSl and ^rP72; C-VSi and BTTl; CNSl and CDCi 7; CNSl and CPi? 7; CiVSi and HSC82; CNSl and &4.R2; CΛ/Si and LHSl; CNSl and Λ/G£i; CNSJ and MPSi 7; CiVSi and NOBl; CNSl and ECMlO; CNSl and S&47; CN1Si and SSA2; CNSl and SS/ϋ; CiVSi and SSA4; CNSl and 5SCi; CNSl and SS£2; CiVSi and SILl; CNSl and SZSi; CNSi and ORMl; CNSl and 0PM2; CNSl and P£Pi; CNSi and PTC2; CNSl and PS£i; CNSi and UBM; CNSl and HΛCi or a truncated intronless HΛCi; CPR3 and CPPd; CPPi and EROl; CPRS and £l/Gi; CPPi and FMOl; CPR3 and HCHl; CPR3 and HSPiO; CPPJ and HSP12: CPR3 and HSP 104; CPR3 and HSP2d; CPΛ3 and HSP 30; CPR3 and HSP42; CPPi and HSP60; CPR3 and HSP75; CPR3 and HSPS2; CPPi and JEMl; CPR3 and MZ)Ji; CPPJ and MDJ2; CPR3 and MPDi; CPR3 and MPD2; CPPi and PDIl; CPR3 and PFDi; CPPJ and ABCl; CPR3 and ,4PJi; CPR3 and ΛTPii; CPPJ and ATPl 2; CPR3 and 5TTi; CPR3 and CDCi7; CPPJ and CPRl: CPR3 and HSCS2; CPPJ and KAR2; CPR3 and IHSi; CPR3 and M?£7; CPΛ3 and MRSH; CPR3 and NOPi; CPPi and ECMlO; CPR3 and _SS47; CPR3 and SS.42; CPPi and SSA3; CPR3 and S&44; CPPi and SSCl; CPR3 and SS£2: CPR3 and SiZi; CPPi and SLSl; CPR3 and OPJlH; CPPi and 0RM2; CPR3 and P£Λ7; CPR3 and PPC2; CPPi and PSEl; CPR3 and [7574; CPR3 and iZ4Ci or a truncated intronless HACl; CPR6 and EROl: CPR6 and £t7G7; CPP 6 and FMOi; CPPd and HCHl; CPR6 and HSPiO; CPP<5 and HSP12; CPR6 and HSP J 04; CPR6 and HSP2d; CPP6 and HSP30; CPR6 and HSP42; CPP<5 and HSP60; CPR6 and HSP7S; CPR6 and HSP52; CPPd and JEMl; CPR6 and MDJi; CPP<5 and MDJ2; CPPd and MPDl; CPR6 and MPD2; CPP 6 and PDH; CPP<5 and PFDl; CPR6 and A8Ci; CPPd and APJl; CPR6 and ΛPPii; CPR6 and ΛPP72; CPPd and BTTl; CP R6 and CDCi 7; CPPd and CPRl; CPR6 and HSC52; CPR6 and /C4P2; CPPd and LHSl: CPR6 and MG£i; CPPd and MRSIl; CPR6 and NOPi; CPR6 and £CM/0; CPΛd and SSAl; CPR6 and SS,42; CPPd and SSA3; CPR6 and S&4^; CPR6 and SSCi; CPPd and SSE2; CP R6 and S727; CPR6 and SZSi; CPPd and
ORMl; CPR6 and 0PM2; CPPd and PERl; CPR6 and PPC2: CP R6 and PS£i; CPR6 and UB14; CPR6 and HACl or a truncated intronless HACh EROl and EUGl; EROl and FMOh EROl and HCHl ; EROl and HSPlO; EROl and HSP 12; EROl and HSP 104: EROl and HSP26; EROl and HSPiO; £7?<9i and HSP42; EROl and HSP60; EROl and HSP7S; £7?<3T and HSP82: EROl and J£MT; EROl and MDJi; £7?07 and MDJ2; EROl and MPD7: £7?O7 and MPD2; EROl and PZ)Ji; EROl and PFDT; £7?<9i and ABCl; EROl and ^PJi; £7?<97 and ATPIl; EROl and Λ7T72; EROl and 73777: £7?O7 and CDC37; EROl and CPi? 7; £i?Oi and HSC82; EROl and 7C47?2; EROl and LHS7; £i?07 and MGEh EROl and MΛSii; £i?0i and NOBl; EROl and TJCMiO; EROl and S&47; £7?<97 and SSA2; EROl and S&43; ER Ol and S&44; £7?0T and SSCl; EROl and 5552; EROl and S7Z7; £7? Oi and SLSl; EROl and ORMi; £7?O7 and 0RM2; EROl and P£7?7; EROl and PIC2; £7? 07 and PSEl; EROl and t/5/4; £7?O7 and HACl or a truncated intronless HACl; EUGl and FM07; EUGl and HCHi; £[7G7 and HSPlO; EUGl and HSP72; £^7Gi and HSP104; EUGl and HSP25; EUGl and HSP30; £t/G7 and HSP 42; EUGl and HSP50; EUGl and HSP 75; £L/Gi and HSP82; EUGl and J£M7; £t/G7 and MDJl; EUGl and MDJ2; EUGl and MPDi; £t/G7 and MPD2; EUGl and PDii; £C/G7 and PFDl; EUGl and v473C7; EUGl and APJ7; /5?7G7 and ATPIl; EUGl and ^PP72; £L/G7 and BTTl; EUGl and CDCi 7; EUGl and CPi? 7; £C/G7 and HSC82; EUGl and 7C4i?2; £f/Gi and ZHS7; £{7G7 and MG757; EUGl and Mi?Sii; £?7Gi and NOBl; EUGl and TiCMiO; EUGl and S&4i; £C7G7 and SSA2; EUGl and 5&43; EI7G7 and SSA4; EUGl and 55C7; EUGl and 557i2; £[/G7 and SILl; EUGl and 5ZS7; £Z7G7 and O7?M7; EUGl and O7?i/2; EUGl and P£7?7: £f/G7 and PTC2; EUGl and P5£7; £C/G7 and UB14; EUGl and H4C7 or a truncated intronless H4C7; FMOl and HCH7; FM07 and HSP70; FMOl and H5P72; FM07 and HSP104; FMOl and HSP26; FMOl and H5P30; FMOi and HSP42; FMOl and H5P60; FM07 and HSP78; FMOl and HSP82; FMOl and J£M7; FM(97 and MDJl; FMOl and A4DJ2; FMOl and MPD7; FM07 and MPD2; FMOl and PD77; FM07 and PFD7; FMOl and ^5Ci; FMOl and _47V7; FM07 and ATPIl; FMOl and ^7T72; FMOl and 5777; FM07 and CDC37; FMOl and CPi?7; FM07 and
HSC82; FMOl and TC47?2; FM07 and LHSl; FMOl and MGEl; FMOl and MRSIl; FMOl and NOBl: FMOl and ECMl O: FMOl and SSAl: FMOl and
5542; FMOl and SSA3; FMOl and SSA4; FMOl and SSCl; FMOl and 55£2;
FMOl and SlLl; FMOl and 5157; FMOi and ORMl: FMOl and 0/?M2; FMOl and P£^;; PM37 and PTC2; FMOl and P5£7; FMO/ and UB14: FMOl and HACl or a truncated intronless HACl: HCHl and HSP/ 0; HCH/ and HSP 12:
HCHl and HSP704; HCHl and H5P26; HCH/ and HSP30; HCHl and 7-/5P^2;
HCH/ and HSP60: HCHl and H5P7S; HCHl and H5P52; HCH/ and JEMl;
HCHl and 7\^DJ7; HCH/ and MDJ2: HCHl and MPD7; HCHl and 71/PD2;
HCH/ and PDIl; HCHl and PFDl; HCH/ and ABCl; HCHl and ΛPJ/; HCHl and ATPIl; HCHl and .47P/2; HCHl and 5777; HCHl and C/3C57; HCH/ and
CPT? 7; HCHl and 775CS2; HCH/ and KAR2: HCHl and IH57; HCHl and
MG7i7; 7/C777 and M7?577; HCHl and /VO57; 77C777 and ECMlO: HCHl and
S&47; HCHl and 55/42; //CH7 and 55,43; HCHl and 55^^; HC//7 and 55C7;
HCHl and 55£2; HCHl and 5/17; i/C/f7 and 5157; HCHl and O7?M7; HC7Y7 and O7?M2; HC7/7 and P£7?7; HCHl and PPC2; //C//7 and P5£7; HCHl and
LΦ/4; HCHl and 7i4C7 or a truncated intronless /£4C7; 7f5P70 and H5P/2;
H5P70 and HSP104: HSPlO and HSP26; HSPlO and HSP30; HSPlO and HSP42;
HSPlO and HSP60: HSPlO and H5P75; H5P70 and HSP82: HSPlO and J£M7;
H5P70 and MDJ7; H5P70 and M73J2; HSPlO and MPD7; H5P70 and MPD2; H5P70 and PD/7; H5P70 and PFD7; 775P70 and A8C7; H5P70 and ^PJ7;
H5P70 and ^7P77; HSPlO and ΛTP72; HSPlO and BTTl; HSPlO and CDC37;
HSPlO and CPR7; HSPlO and H5CS2; H5P70 and KAR2; HSPlO and LHSl;
HSPlO and MG£7; H5P70 and MRSIl; HSPlO and NOBl; HSPlO and £CM70;
HSPlO and SSAl; HSPlO and SSA2; HSPlO and 55Λ3; HSPlO and 55Λ4; 7/5P70 and 55C7; HSPlO and 55£2; H5P70 and 5/17; HSPlO and 515/; HSPlO and
O7W/7; //5P70 and 0RM2; HSPlO and P£7?7; HSPlO and P7T2; //5P70 and
P5£7; HSPlO and OB/4; H5P/0 and HACl or a truncated intronless HACl;
HSP12 and HSP104; HSP12 and Z/5P26; //5P/2 and HSP30; HSPl 2 and //5P¥2;
775P72 and HSP60: HSP12 and //5P75; HSP12 and //5PS2; //5P72 and JEMl; H5P/2 and M/)J7; HSP 12 and MDJ2; 7/5P/2 and MPDl; HSP 12 and MP£>2;
//5P72 and PDIl; HSP 12 and PFD/ ; HSP 12 and Λ/3C7; //5P/2 and .4PJ/ ; HSP12 and ATP11; HSP12 and ATP12; HSP12 and BTT1: HSP12 and CDC37; HSP 12 and CPR7; HSP 12 and HSC82; HSP 12 and KAR2: HSP 12 and LHS1; HSP 12 and MGE1; HSP 12 and MRS11; HSP 12 and NOB1; HSP 12 and ECM10; HSP 12 and SSA1; HSP12 and SSA2; HSP 12 and SSA3; HSP 12 and SSA4; HSP12 and SSC1; HSP12 and SSE2; HSP 12 and SIL1: HSP12 and SLS1; HSP 12 and ORM1; HSP 12 and ORM2; HSP 12 and PER1; HSP12 and PTC2; HSP 12 and PSE1; HSP12 and UBI4; HSP12 and HAC1 or a truncated intronless HAC1; HSP 104 and HSP26; HSP 104 and HSP 30; HSP 104 and HSP42; HSP 104 and HSP60; HSP104 and HSP78; HSP104 and HSP82; HSP104 and JEM1; HSP104 and MDJ1; HSP 104 and MDJ2; HSP104 and MPD1; HSP104 and MPD2; HSP104 and PDI1; HSP104 and PFD1; HSP 104 and ABC1; HSP104 and APJ1; HSP 104 and ATP11; HSP104 and ATP 12; HSP104 and BTT1; HSP104 and CDC37: HSP 104 and CPR 7; HSP104 and HSC82; HSP 104 and KAR2; HSP 104 and I//57: fl£P704 and MG£7; 7J5P704 and MRSIl; HSP 104 and NO57; HSP 104 and 7SCM7O; H5P70^ and SSAl; HSP 104 and 55^2; H5P70^ and SSA3; HSP 104 and 5&44; HSP 104 and 5SC7; HSP 104 and SS£2; HSP 104 and 5717; 775P7^ and 5157; H5P704 and (97?M7; HSP 104 and ORM2; //5P70^ and PERl; HSP104 and P7C2; 775P70^ and PSEl; HSP 104 and C/B/4; HSP104 and /i4C7 or a truncated intronless 7£4C7; HSP26 and 775P50; HSP26 and H5P42; 775P26 and HSP60; HSP26 and H5P7S; HSP26 and 7J5P52; H5P2d and JEMl; HSP26 and AfDJi; 7f5P2d and MDJ2; HSP26 and MPD7; HSP26 and A£PD2; H5P25 and PD/7; HSP 26 and PF737; H5P26 and AB Cl; HSP 26 and ΛPJ7; HSP 26 and ^rP77; 775P25 and ^7T72; HSP26 and 73777; HSP26 and CDC57; H5P2(5 and CPT? 7; HSP26 and H5C52; 775P25 and TC4T?2: HSP26 and Z//57; HSP26 and MG£7; 7/5P25 and M7?577; HSP26 and NO737; HSP 26 and £CM70; 7J5P2d and SSAl; HSP26 and SS42; H5P25 and 55^43; HSP26 and SS^^; HSP26 and 55C7; 775P25 and 55£2; HSP 26 and 5717; 775P25 and 5L57; HSP26 and O7?i/7; HSP 26 and OT?M2; H5P2(5 and P£7?7; HSP26 and P7C2; 7/5P25 and P5£7; 775P25 and UBU; HSP26 and 7£4Ci or a truncated intronless HACl; HSP30 and HSP42; JΪSP30 and /75P50; HSP 30 anfr HSP78; HSP30 and 7/5P52; HSP 30 and J£M7;
T75P30 and MDJl; HSP30 and MDJ2; T75P30 and AfPDi; HSP30 and AfPD2; HSP30 and PDIl; HSP 30 and PFDJ: HSP 30 and ABCl; HSP30 and APJl: HSP30 and ATPIh. HSP30 and ATPJ 2; HSP 30 and BTTJ: HSP30 and CDC37: HSP 30 and CPi? 7; HSP 30 and 775CS2; //5PiO and KAR2: HSP 30 and ZJ/Si; HSP30 and MGEJ: HSP 30 and M7?577: HSP 30 and /VO/37; /f5P30 and ECMJO: HSP30 and 55Λ7; HSP30 and 5542; //5PJO and SSA3; HSP30 and SS/44; //5PiO and 55C7; HSP 30 and SSE2; HSP30 and SILJ: HSP30 and 5L57; HSP30 and OΛM7; //5PiO and ORM2; HSP 30 and P/£Z?7; //5PiO and PTC2\ HSP 30 and P5£7; HSP30 and £//3/4; //5PiO and HACJ or a truncated intronless HACJ: HSP 42 and //5PdO; //5P42 and HSP 78; HSP 42 and /75PS2; HSP 42 and J£M/: //5P42 and MDJJ: HSP 42 and MDJ2; HSP 42 and MPDi; //5P42 and MPD2; HSP 42 and PD/7; //5P42 and PFDJ; HSP 42 and .4/3C7; HSP 42 and APJ/; //5P42 and ATPJI; HSP42 and Λ7P/2; /75P42 and BTTJ: HSP42 and CDCi 7; HSP 42 and CPi? 7; //5P42 and HSC82; HSP42 and /ατ?2; //5P42 and LHSl \ HSP 42 and MGiSi; HSP 42 and M7?57/; //5P42 and NOBl; HSP 42 and ECMJO: 7/5P42 and 55Λi; i/5P42 and SSA2; HSP42 and 55^i; //5P42 and SSA4: HSP42 and 55Ci; HSP 42 and 55£2; //5P^2 and SILl; HSP 42 and 515/; //5P42 and ORMJ; HSP 42 and 0ΛM2; HSP 42 and P£/?/; H5P^2 and PTC2; HSP 42 and P5£7; //5P42 and UBI4; HSP42 and /i4C/ or a truncated intronless //4Ci; HSP60 and //5P 75; //5P60 and HSP82; HSP 60 and J£M7; //5P60 and MDJ/; HSP 60 and MDJ2; HSP 60 and MPDi; //5P60 and MPD2; HSP 60 and PD//; HSP 60 and PFDi; //5PdO and A8C7; HSP 60 and APJ7; //5PdO and ATPIJ; HSP60 and Λ7Pi2; HSP 60 and /5777; //5PdO and CDC31; HSP 60 and CPi? 7; HSP60 and //5CS2; H5Pd0 and KAR2; HSP 60 and ZJ/57; //5PdO and MGEl; HSP 60 and Λ/ΛS77; HSP 60 and NOS/ ; //5PdO and ECMJO; HSP 60 and 554/; HSP60 and 5542; 7/5PdO and 554i; HSP60 and 55Λ4; //5PdO and 55C/; HSP60 and 55£2; HSP60 and 5/Z7; //5PdO and 515/; HSP60 and <9i?Mi; //5PdO and 0RM2; HSP 60 and P£i?i; HSP60 and P7C2; //5PdO and P5£Z; HSP60 and L/73/^; //5PdO and HACl or a truncated intronless HACl; HSP78 and /75P52; HSP78 and JEM7; /75P75 and MDJl; HSP78 and MDJ2; 775P75 and MPD7; HSP 78 and MPD2; HSP 78 and PD/7; HSP 78 and PFD7; //5P 78 and Λ/3C/;
//5P75 and ΛPJ7; HSP78 and ΛZP77; //5P7S and Λ7P72; HSP78 and 7377/; HSP78 and CDC37: HSP78 and CPR7; HSP 78 and HSC82: HSP78 and KAR2; HSP 78 and LHSl: HSP 78 and MGEh HSP 78 and MRSIl; HSP78 and NOBl: HSP78 and £CAfi0; //5P 75 and SSAl: HSP78 and S&42; HSP 78 and 5SΛ3; /75P7S and SSA4; HSP78 and 55Ci; 7/5P7,§ and SSE2; HSP78 and 5717; HSP78 and 5L57; /75P7S and ORMl: HSP78 and ORM2; HSP78 and />£/?/; 77SP75 and PTC2; HSP78 and P5£7; 7/5P7S and UBI4; HSP78 and /£4C7 or a truncated intronless U4C7; HSP82 and JEAf/; 7/5PS2 and MDJ7; HSP82 and A£DJ2; H5PS2 and MPDl: HSP 82 and AfPD2; HSP 82 and P7377; 775P52 and PFDl; HSP82 and /15C7; /75PS2 and APJl; HSP82 and ATPIl; HSP82 and ATP 12; HSP82 and BTTl; HSP82 and CDC37; HSP82 and CPR7; HSP82 and HSC82; HSP82 and 7C4Λ2; 7/5PS2 and LHSl: HSP82 and MG£7; 77SP52 and MRSIl; HSP82 and NOBl: HSP82 and £CM70; 7KP52 and 55^7; 775P52 and SSA2; HSP82 and SSA3: HSP82 and SSA4; HSP82 and S5C7; /75PS2 and SSE2: HSP82 and 57Z7; HSP82 and 5Z57; 7/SPS2 and ORMl; HSP82 and OKM2; H5PS2 and PERl ; H5PS2 and PTC2; HSP82 and P57i7 ; 77SPS2 and UBI4; HSP82 and HACl or a truncated intronless 7i4C7; JEMl and AfDJi; J£M7 and MDJ2; JEMl and MPDl; JEMl and MP732; J73M7 and PD77; J£M7 and PFDl; JEMl and ABCl; JEMl and APJ7; J£Mi and ATPIl; JEMl and ^TP72; JEMl and 5777; JEMl and CDC57; JTiMi and CPR7; JEMl and 775C52; JEM7 and KAR2; JEMl and I77S7; J75M7 and MGEl; JEMl and MRSIl; JEMl and 7VO757; JEMl and ECMlO; JEMl and S&47; J£M7 and SS42; J£M7 and SSA3; JEMl and 55L44; J£M7 and SSCl; JEMl and SSE2; JEMl and 5/17; J£M/ and SLSl; JEMl and ORMi; JEM7 and ORΛ42; JEMl and PERl; JEMl and PTC2; J£M7 and PSEl; JEMl and [/574; J£M7 and HACl or a truncated intronless HACl; MDJl and MDJ2; MDJl and MPD7; AfDJi and MP732; MDJl and P7577; AfDJi and PFDl; MDJl and ABCl; MDJl and ΛPJ7: MDJ7 and Λ7P77; AfDJi and ^7P72; M73J7 and BTTl; MDJl and CDC37; MDJl and CPR7; MDJl and HSC82; MDJl and 7Oi7?2; AfDJi and LHSl; MDJl and MGEl; MDJl and M?S77; AfDJi and NOBl; MDJl and £CAfi0; AfDJ/ and 55,47; AfDJi and 5&42; MDJl and SSA3; MDJl and 55Λ4; AfDJi and 55C7; M73J7 and 55£2; MDJl and SILl; MDJl and 5157;
AfDJi and ORMl; MDJl and 0RM2; MDJl and P£7?7; AfDJ/ and PTC2; MDJl and PSEl: MDJl and UB14; MDJl and HACl or a truncated intronless HACl: MDJ2 and MPDl; MDJ2 and MPD2: MDJ2 and PDIJ; MDJ2 and PFDl: MDJ2 and ,4P>C7; MDJ2 and _4PJ7; Mλ/2 and ATPIl: MDJ2 and .477V 2; MDJ2 and BTTl; MDJ2 and CDC37; MDJ2 and CPi? 7; MDJ2 and HSC82: MDJ2 and /C4T?2; MDJ2 and Z//5T ; MDJ2 and MG£7 ; MDJ2 and MRSl 1 ; MDJ2 and NOBl : MDJ2 and ECMlO: MDJ2 and SSAl: MDJ2 and SSA2: MDJ2 and 5&43; MDJ2 and S&44; MDJ2 and SSCl; MDJ2 and 55£2; M73J2 and SILI; MDJ2 and 5LST; MDJ2 and 0T?MT; MDJ2 and 0RM2; MDJ2 and P£T?7; MDJ2 and PTC2: MDJ2 and P5£T; MDJ2 and f/T3/4; MDJ2 and HACl or a truncated intronless HACl; MPDT and MPD2; MPDl and PD/T ; MPDl and PP1DT ; MPDT and ABCl ; MPDT and APJl: MPDl and ATPIl; MPDl and ATP 12; MPDl and 57T7; MPDl and CDC37; MPD7 and CPT? 7; MPDl and 77SC52; MP7)7 and KAR2; MPDl and 17/57; MPD7 and MG£7; MPDl and MRSIl; MPDl and Mλδ7; MPD7 and £CM76>; MPDl and 55.47; MPDl and 55,42; MPD7 and 55^3; MPD7 and SSA4: MPDl and 55C7 : MPDl and 55£2; MP737 and 5/Z7 ; MPD7 and 5157 ; MPDl and ORMl: MPDl and 0RM2; MPDl and P£7?7; MPDl and PTC2; MP£>7 and P5£7; MPD7 and UBI4; MPDl and HACl or a truncated intronless /£4C7; MPD2 and PD/7; MPD2 and PFD7; MPD2 and ABCl; MPD2 and ^PJ7; MPD2 and ΛIP77; MPD2 and ^PP72; MPD2 and 5777; MP732 and CDC37; MPD2 and CPT? 7; MP732 and H5CS2; MPD2 and 7C4T?2; MP732 and LHSl; MPD2 and MG£7; MPD2 and MT?577; MPD2 and NO737; A4PD2 and ECMlO; MPD2 and 5547; MP£>2 and 55Λ2; MPD2 and 55^3; MPD2 and 55Λ4; MPD2 and 55C7; MPD2 and 55£2; MPZ)2 and 5/Z7; MPD2 and 5157; MPD2 and ORMT; MP752 and 0RM2; MPD2 and P£7?7; 7l^P/32 and PTC2; MP D2 and P5£7; MPD2 and E/B/4; MPD2 and HACl or a truncated intronless /i4C7; PZ)/7 and PFDT; P£>/7 and ^73C7; PDTT and ΛPJ7; PD77 and y4rP77; PD/7 and ATP 12; PDIl and T37TT; PDIl and CDC37; PDIl and CPT? 7; PDIl and /75CS2; PDIl and KAR2; PDIl and I//57; PD/7 and MGEl: PDIl and M7?577; PDIl and M95T; PD/7 and ECMlO; PDIl and 55Λ7; PD/7 and 5542; PD/7 and SSA3; PDIl and SSA4; PDIl and 55C7; PD/7 and SSE2; PDIl and 5/ZT; PDIl and 515/; PD/7 and 07?jrø; PD/7 and ORI42; PDIl and P£T?T; PDTT and PTC2\ PDIl and P5£T; PDIl and UB14; PDIl and HACl or a truncated intronless HACl: PFDl and ABCl \ PFDl and APJl: PFDl and ATPIl: PFDl and ATPl 2: PFDl and BTTl: PFDl and CDC57; PFZ); and CPRl: PFDl and //5CS2; PFDl and 7C4T?2: PFDi and LHSl: PFDl and MG£i; PFD7 and MRSIl: PFDl and /VOTiT; PFDl and FCMTO; PFDT and SSAl; PFDl and 55,42; PFDl and 5&45; PFDT and SSA4: PFDl and 55CT; PFDT and SSE2: PFDl and 5/17; PFD7 and SLSl: PFDl and ORMl: PFDl and O7ΪM2; PFD7 and PERl; PFDT and PTC2; PFDl and P5FT; PFD7 and OBU: PFDl and HACl or a truncated intronless /14CT; ,45CT and ΛPJ7; ABCl and ,4 FP/ 7; ABCl and ATPl 2: ABCl and BTTl; ABCl and CDC57; ,4SCT and CPT? 7; ABCl and //5CS2; ,45CT and KAR2; ABCl and 1T/5T; ,45CT and MGFT; ABCl and MRSIl; ABCl and M957; ,45CT and ECMlO; ABCl and 5&4T; ,45CT and SSA2: ABCl and SSA3; ABCl and 5-5-4-/; ,45CT and SSCl; ABCl and 55F2; ABCl and SILl; ABCl and 5157; Λ5C7 and O#M7; ^5C7 and O7ΪM2; ^P>C7 and PF7?7; ^73C7 and PTC2; ABCl and P5F7; ^P,C7 and UBM; ABCl and 7i4C7 or a truncated intronless HACl; APJl and Λ7P77; ΛPJ7 and ^TP72; ^PJ7 and BTTl; APJl and CDC37; APJl and CPT? 7: ^PJ7 and HSC82; APJl and KAR2; APJl and LHSl; APJl and MGEl; APJl and MRSIl: APJl and /VO57; ^PJ7 and ECMlO; APJl and S&47; ^PJ7 and 55^2; ^PJ7 and SSA3; APJl and SSA4; APJl and S5C7; APJl and SSE2; APJl and 5717; ^PJ7 and 5157; v4PJ7 and ORMl: APJl and 0RM2; APJl and P£T?T; ^PJT and PTC2; APJl and P5F7; ^PJ7 and UBU; APJl and HACl or a truncated intronless T14C7; ^7P77 and ΛFP72; ATPIl and 75PH; Λ7P77 and CDC37; ATPIl and CPT?7; ^TP77 and H5C52; ^TP77 and KAR2; ATPIl and LHSl; ATPIl and MGEl; ATPIl and M7?577; ATPIl and NO7i7; ^7P77 and ECMlO; ATPIl and 55,47; Λ7P77 and 55^2; ^TP77 and 55,43; ^rP77 and SSA4; ATPIl and 55C7; Λ7P77 and 55F2; Λ7P77 and 5/17; ^rP77 and 5157; ATPIl and ORMl; ATPIl and 0RM2; ATPIl and PERl; ATPIl and PTC2; ATPIl and P5F7; ATPIl and LΦ/^; ATPIl and 714C7 or a truncated intronless HACl; ATP12 and T37Y7; ΛTPT2 and CDC37: ATP12 and CPR7; ATP 12 and T/5C52; Λ7P72 and KAR2; ATPl 2 and LHSl; ATP 12 and MGF7; ATP12 and M7?577; Λ7P72 and NOBl; ATP12 and FCM70; ^PP72 and
SSAl; ATP12 and 55Λ2; Λ7P72 and SSA3; ATP12 and 55.44; ^PP72 and 55C7; ATP] 2 and SSE2: ATPl 2 and SILJ: ATP 12 and SLSl: ATP12 and ORMl-, ATP12 and ORM2; ATP12 and PERl: ATP12 and PTC2: ATP12 and PSEl; ATP12 and £7574; -4.TP72 and HACl or a truncated intronless HACl; BTTl and CDCJ7; BTTl and CPi? 7; 5117 and HSC82; BTTl and 7C4T?2: 5777 and LHSl; BTTl and MGEl; BTTl and M?577; 73777 and NOBl; BTTl and ECMlO; BTTl and 5547; 5777 and SSA2; BTTl and S5L43; 73777 and SSA4: BTTl and 55C7; 73777 and SSE2; BTTl and SILl; BTTl and 5L57: 73777 and ORMl; BTTl and ORM2; 5777 and PERl; BTTl and P7C2; 5777 and PSEl; BTTl and UBM; BTTl and 7I4C7 or a truncated intronless 7i4C7; CDC37 and CPT? 7; CZ)CJ 7 and HSC82: CDC37 and 7£4T?2; CT)CJ 7 and Z/T57; CDCJ 7 and MGEl; CDC37 and MT?5T7; CDC37 and 7VO57; CT)CJ7 and ECMlO; CDC37 and 55.47; C7)CJ7 and SSA2; CDC37 and SSA3; CDC37 and SSA4; CDC37 and 55CT; CDC37 and 55£2; CDCJ 7 and 5717; CDC37 and 5157; CDC37 and ORMT; CDCJ 7 and 0RM2; CDC37 and P£T?T; CDCJ 7 and P7C2; CDC 37 and P5£T; CDC 37 and [/BW; CDCJ 7 and HACl or a truncated intronless HACl; CPR7 and 775CS2; CPT?7 and KAR2; CPR7 and 17757; CPT? 7 and MGTfT; CPT? 7 and MRSIl; CPR7 and NOBl; CPR7 and ECMlO; CPR7 and SSAl; CPRl and 55,42; CPT?7 and SSA3; CPT?7 and 55,44; CPT?7 and 55C7; CPT? 7 and 55£2; CPT? 7 and 57IT; CPT? 7 and 5I5T; CPT? 7 and OT?M7; CPT? 7 and <9T?M?; CPT? 7 and P£T?7; CPT? 7 and P7C2; CPT? 7 and P5£T; CPT? 7 and UBI4; CPT?7 and 7£4C7 or a truncated intronless HACl; HSC82 and TC4T?2; HSC82 and L7757; HSC82 and MGEl; HSC82 and M7?577; 775C52 and NOBl; HSC82 and ECMlO; HSC82 and 5547; HSC82 and 55Λ2; 775CS2 and SSA3; HSC82 and SSA4; HSC82 and 55C7; 77SCS2 and 55£2; H5C52 and 5TI7; T75C52 and 57,57; HSC82 and OT?MT; H5CS2 and OT?M2; 775C52 and PTiT?T: 775C52 and P7C2; 775CS2 and P5£T; HSC82 and VB14; HSC82 and TΪ4C7 or a truncated intronless 7i4C7; 7C4T?2 and I775T; T<^T?2 and MGTiT; KAR2 and M?577; 7C4T?2 and NOBl; KAR2 and ECMlO; KAR2 and 55ΛT; 7C4T?2 and SSA2; KAR2 and 55ΛJ; 7C47?2 and SSA4; KAR2 and 55C7; 7£4T?2 and 55£2; TC4T?2 and 57Z7; TαT?2 and 5Z5T; 7C47?2 and OT^T; TL4T?2 and OT?M2; TC4T?2 and P£T?7; TC47?2 and P7C2; 7C47?2 and P5£T; KAR2 and UBI4; KAR2 and 77ΛC7 or a truncated intronless 77ΛC7; 17757 and MG£T; 1T757 and MRSIl; LHSl and NOBl; LHSl and ECMlO; LHSl and SSA]: LHSJ and 5542: LHSl and 5543: LHSl and 5544; LHSl and 55C7; LHSl and 55£2; LHSl and SILl \ LHSl and SLSl \ LHSl and ORMl: LHSl and ORM2; LHSl and P£R/: /,//57 and PTC2\ LHSl and P5£/; LHSl and t/B/4; 1/75/ and HACl or a truncated intronless HACl; MGEl and M7Ϊ5/7; Λ/G£7 and NOBl: MGEl and ECMlO: MGEl and 554/; MG£/ and 5542; MGEl and S&43; MGEl and 5544; MG£/ and SSCl; MGEl and 55£2; MG£7 and SILl; MGEl and 515/; MGEl and ORJW7; MG£/ and 0RM2; MGEl and P£R/: MG£7 and PTC2; MGEl and P5£7; MGEl and rø/4: MG£7 and HACl or a truncated intronless HACl; MRSIl and NOBl; MRSIl and ECMlO; MRSIl and 5547; M7?57/ and SSA2; AdRSIl and SSA3; MRSIl and 5544; M/?57/ and SSCl; MRSIl and 551^: MRSIl and 5/17; M7£S77 and SLSl; MRSIl and 07^1/7; MΛSV/ and 0RM2; MRSIl and P£/?7; MRSIl and P7T2; A/RS77 and PSEl; MRSIl and L/B/4; MR577 and HA Cl or a truncated intronless 7Ϊ4C7; NOBl and ECMlO; NOBl and SX47; NO57 and SSA2; NOBl and 55L43; NOBl and 55^4; NO57 and 55C7; NOBl and 55£2; NOBl and S/Z7; NO57 and SLSl; NOBl and O7JM7: NO57 and 0RM2; NOBl and P£7?7; NOBl and P7C2; NO57 and P5£7; NOBl and C/574; JVOP.7 and 7Ϊ4C7 or a truncated intronless HACl: ECMlO and 5&47; ECMlO and 55^2; £CM70 and SSA3: ECMlO and 5&44; £CM7(9 and SSCl; ECMlO and SS£2; £CM70 and 5/17; £CM70 and SLSl; ECMlO and ORMl; ECMlO and O7LM2; £CM70 and P£7?7; ECMlO and PPC2; £CM76» and P5£7; ECMlO and C/5/^; £CM70 and HACl or a truncated intronless HACl; SSAl and SSA2; SSAl and S&43; 5Sv47 and SSA4; SSAl and 55C7; SSAl and SSE2; 55L47 and SILl; SSAl and 5I57; 55^7 and O7?M7; SSAl and O7?M2; SSAl and P££7; 5SL47 and PTC2; SSAl and P5£7; SS47 and UBI4; SSAl and 7i4C/ or a truncated intronless /£4C7; 55^2 and S5>43: 55^2 and SSA4; SSA2 and 55C/; SSA2 and 55£2; 5&42 and SILl ; SSA2 and 5157 ; 55,42 and ORMl; SSA2 and O7?M2; SSA2 and P£7?7; 55^2 and PTC2; SSA2 and P5£7; 55,42 and UBH; 5542 and HACl or a truncated intronless HACl; SSA3 and 554-/; 5543 and 55C/; SSA 3 and 55£2; 5543 and 5/Z7; SSA3 and 5X57; SSA3 and OΛAf7; 5543 and 0RM2; SSA3 and P£Λ/; SSA3 and P7T2; 55/13 and P5£7; 55^3 and VB14; SSA3 and HACl or a truncated intronless HACl;
SSA4 and 55C7; SSA4 and 55£2; 5544 and SlLl; SSA4 and 5157; SSA4 and ORMl; SSA4 and ORM2; SSA4 and PER]: SSA4 and PTC2; SSA4 and PSEl: SSA4 and UBU; SSA4 and HACl or a truncated intronless HACl: SSCl and SSE2; SSCl and SILl: SSCl and 5157; SSCl and 0ΛM7; 55Ci and 0RM2; SSCl and P£T?/; 55C7 and PTC2: SSCl and P5£7; SSCl and [/T3/4: 55C/ and HACl or a truncated intronless 7YΛC7; 55£2 and 5/Z7; SSE2 and 5157; 55£2 and ORMl; SSE2 and 0TW2; 55752 and PERl: SSE2 and P7C2; 55£2 and PSEl \ SSE2 and UBI4; SSE2 and 77ΛC7 or a truncated intronless /7ΛCT; 5/17 and 5157; SILl and ORMl; 5/17 and 0RM2; SILl and PERl \ SILl and PTC2; SILl and P5£/: SILl and UBI4; SILl and HACl or a truncated intronless HACl: SLSl and ORMl: SLSl and <9#M2; 5157 and PERl; SLSl and PTC2: SLSl and P5£7; 5157 and UBU; SLSl and T7ΛC7 or a truncated intronless /i4C7; ORMl and OT?M2; 0T?M/ and PERl; ORMl and P7T2; 6>£M7 and PSEl; ORMl and E/B/4; ORMl and /i4C/ or a truncated intronless HACl; 0RM2 and PERl; 0RM2 and P7C2; O7^M2 and P5£7; 0RM2 and f/73/^; ORM2 and HACl or a truncated intronless /Ϊ4C7; PERl and PTC2; PERl and P5£7; PERl and LΦ/¥; P£7?/ and 7£4C/ or a truncated intronless HACl; PTC2 and P5£7; PTC2 and UBU; PTC2 and U4C7 or a truncated intronless 7Ϊ4C/; P5£7 and UBU; PSEl and /£4C7 or a truncated intronless HACl: UBU and HACl or a truncated intronless /£4C7/ 77M9 and Λ/£4/; r/M9 and CCP2; TIM9 and CCH; 77M9 and CCT4; T1M9 and CCrJ; TIM9 and CC7tf; HMP and CCTl; TIM9 and CC7S; T/MP and CNSl; TIM9 and CP7?3; TIM9 and CPTW; T/MP and ER 01; TIM9 and £[/G7; TIM9 and FM07 ; 77MP and HCHl; TIM9 and H5P70; 7YMP and HSPl 2; TIM9 and HSP104; TIM9 and /75P26; 77MP and HSP30; TIM9 and /75P42; 77MP and HSP60; TIM9 and /f5P75; TIM9 and /75PS2; 77MP and JTfM/; TIM9 and MDJ7; 77MP and MDJ2; r/MP and MPDl; TIM9 and MP732; 77MP and PDIl; TIM9 and PF737; TIM9 and ^5C7; r/MP and ΛPJ/; TIM9 and ^PP//; 7/MP and Λ7P72; TIM9 and 5PH; TIM9 and CXO 7; 77MP and CPT? 7; TIM9 and T75CS2; T/MP and T£4T?2; T7MP and LHSl; TIM9 and MGEl; TIM9 and MT?5T/; TIM9 and /VOTi/; 77MP and ECMlO; TIM9 and 5&47; TIM9 and 55Λ2; 7YMP and SSA3; TIM9 and 55^4; r/MP and 55C7; TIM9 and 55£2; TIM9 and 5/1/; 77MP and 57L57; 77MP and ORMl;
TIM9 and 0RM2; TIM9 and P£7?7; TIM9 and P7O; 77MP and P5£/; T1M9 and UBI4; TIM9 and HACJ or a truncated intronless HACJ: PAMl 8 and AHAl; P AM 18 and CCT2: PAMJ 8 and CCT3: P AM 18 and CCT 4\ PAMJ 8 and CC75; PAMl 8 and CC76; PAMJ 8 and CCT7; PΛMiS and (XTS; P^MiS and CΛ'S7; PyIMiS and CPR3; PAM18 and CP/?6: PAM18 and £POi; P/1M7S and EUGl; PyIMiS and PMOi; PAM18 and HCHi; PyIMiS and HSPlO: PAMJ8 and HSPi2; PyIMiS and HSPJ04; PAMJ8 and HSP26; PAMJ8 and HSP30; PyIMiS and HSP42; PAM18 and HSP60; PyIMiS and HSP78: PAMJ8 and HSPS2; PAM18 and JZM/ ; PΛMiS and MDJl; PAMl 8 and Affl>J2; PΛM/S and MPDl: P AMI 8 and MP/J>2; PAM18 and PDH; PΛMiS and PFDl; PAM18 and yϊPCi; A4M7S and APJl; PAM18 and ATPIl; PAM18 and /f7Pi2; PyIMiS and BTTl; PAM18 and CZ)Ci 7; PAM18 and CPi? 7: PyIMiS and HSC82; PAMl 8 and ZC4P2; PyIMiS and LHSl; PAM18 and MG£7; PAM18 and Mi?Sii; PyIMiS and NOBl; PAM18 and ECMiO; PyiMiS and SSAl; PAM18 and S&42; PAM18 and S&43; PΛMiS and SSA4; PAM18 and SSCi; PyIMiS and SSE2; PAM18 and 5/17; PAM18 and 51ISi; PyIMiS and ORMl; PAM18 and ORM2; PAM18 and P£i?i; PyIMiS and PTC2; PAMl 8 and PSiii; PyIMiS and UBI4; PAMl 8 and /£4C/ or a truncated intronless HyICi; TCPl and ΛHΛi, 7CPi and CCT2; TCPl and CCT; TCPi and CCT4; TCPl and CCPi; 7CPi and CCT6; TCPl and CCTl; TCPl and CCPS; 7CP7 and CNSl; TCPl and CPPJ: TCPJ and CPR6; TCPl and EROl; TCPl and ZC/G7; TCP7 and FMOl; TCPl and HCHl; TCPl and HSPiO; PCPi and HSP12; TCPl and HSPi 0¥; TCPJ and HSP25; 7CPi and HSP30; TCPJ and HSP42; 7CPi and HSP 60; TCPJ and HSP 78; TCPJ and HSPS2; 7CPi and JEMJ; TCPJ and MDJ/; 7CP7 and MDJ2; TCPJ and MPDl; TCPl and MPZ)2; 7CP7 and PDIl; TCPl and PFDi; 7CPi and ABCl; TCPl and APJl; TCPl and Λ7Pii; 7CPi and y*7Pi2; 7CPi and 577i; TCPl and CDC37; 7CPi and CPR7; TCPl and HSCS2; 7CPi and KAR2; TCP! and LHSl; TCPl and MGZ/; 7CPi and MRSIl; TCPl and .VOJ?/; 7CPi and ECMlO; TCPl and SSAl; TCPl and SS42; 7CPi and SSA3; TCPl and SSΛ4; 7CPi and SSCl; TCPl and SSE2; TCP! and S7I7; 7CP7 and S1S7; 7CPi and ORMl; TCPl and ORlvl2; TCPl and PERl; TCPl and P7C2; 7CP7 and PS£7; 7CP7 and UBU; TCPJ and HACJ or a truncated intronless H4C7; TIM9 and PAMI 8; TM 9 and 7CPi; or PAMJ 8 and 7CPi. The first, second and third recombinant genes may, or may not. each individually be present on a plasmid within the host cell (which may, or may not, be a 2μm- family plasmid, as discussed above) or be chromosomally integrated within the genome of the host cell. It will be appreciated that any combination of plasmid and chromosomally integrated first, second and third recombinant genes may be used. For example, the first, second and third recombinant genes may, or may not, each individually be present on a plasmid, and this may, or may not, be either the same plasmid or different plasmids. Alternatively, the first recombinant gene may, or may not, be present on a plasmid, and second and third recombinant genes may, or may not, be chromosomally integrated within the genome of the host cell. Alternatively, the first and second recombinant genes may. or may not, be present on a plasmid and the third recombinant gene may, or may not, be chromosomally integrated within the genome of the host cell. Alternatively, the first and third recombinant genes may, or may not, be present on a plasmid and the second recombinant gene may, or may not, be chromosomally integrated within the genome of the host cell. Alternatively, the first and second recombinant gene may, or may not. be chromosomally integrated within the genome of the host cell and the third recombinant gene may, or may not, be present on a plasmid. Alternatively, the first, second and third recombinant genes may, or may not, each individually be chromosomally integrated within the genome of the host cell.
Plasmids used for this purpose may, or may not, be plasmids, such as 2μm-family plasmids, as defined below. Thus, in one embodiment, a method according to the first aspect of the invention does not involve a host cell in which the first, second and third recombinant genes are all present on the 2μm-family plasmid.
Accordingly, as a second aspect, the present invention also provides a plasmid wherein the plasmid comprises two different genes (the first and second recombinant genes) encoding different chaperones. In one preferred embodiment, the plasmid may. or may not. further comprise a gene encoding a heterologous protein (the third recombinant gene), such as a heterologous protein as described above. A plasmid according to the second aspect of the invention may, or may not, be a 2μm-family plasmid.
A third aspect of the present invention provides for the use of the plasmid of the second aspect of the invention as an expression vector to increase the production of a desired protein, including as heterologous protein, such as a fungal (optionally yeast) or vertebrate protein. The desired protein may, or may not, be encoded by a recombinant gene that is present as part of the plasmid, or present in the host cell on a different plasmid, or present in the host cell as a transgene that is integrated in the host cell's chromosome.
A fourth aspect of the invention provides a host cell comprising a plasmid as defined above. The host cell may, or may not, further comprise a recombinant gene encoding a desired heterologous protein. Where the recombinant gene that encodes the desired heterologous protein (the "third recombinant gene") is not present as part of the same plasmid that encodes the first and second chaperones, then the host cell may, or may, not, comprise the third recombinant gene on a different plasmid, or as a transgene that is integrated in the host cell's chromosome.
As a fifth aspect, the present invention provides a host cell which comprises the first, second and third recombinant genes. The first, second and third recombinant genes may, or may not, each individually be present on a plasmid within the host cell (which may, or may not, be a 2μm-family plasmid, as discussed above) or be chromosomally integrated within the genome of the host cell. It will be appreciated that any combination of plasmid and chromosomally integrated first, second and third recombinant genes may be used., as discussed above. Thus, the host cell may, or may not, comprise the first, second and third recombinant genes each individually present on a plasmid, and this may, or may not. be either the same plasmid or different plasmids. Alternatively, the host cell may. or may not, comprise the first recombinant gene on a plasmid, and second and third recombinant genes chromosomally integrated within the genome of the host cell. Alternatively, the host cell may, or may not, comprise the first and second recombinant genes on a plasmid and the third recombinant gene chromosomally integrated within the genome of the host cell. Alternatively, the host cell may. or may not, comprise the first and third recombinant genes on a plasmid and the second recombinant gene chromosomally integrated within the genome of the host cell. Alternatively, the host cell may, or may not, comprise the first and second recombinant genes chromosomally integrated within the genome of the host cell and the third recombinant gene present on a plasmid. Alternatively, the host cell may, or may not, comprise the first, second and third recombinant genes each individually chromosomally integrated within the genome of the host cell.
The 2uni-familv plasmids:
For the purposes of the present invention, a plasmid may, or may not, be a 2μm- family plasmid. Certain closely related species of budding yeast have been shown to contain naturally occurring circular double stranded DNA plasmids. These plasmids, collectively termed 2μm-family plasmids, include pSRl, pSB3 and pSB4 from Zygosaccharomyces roiaii (formerly classified as Zygosaccharomyces bisporus), plasmids pSBl and pSB2 from Zygosaccharomyces bailii, plasmid pSMl from Zygosaccharomyces ferment ati, plasmid pKDl from Kluyveromyces drosphilarum, an un-named plasmid from Pichia membranaefaciens (hereinafter "pPMl") and the 2μm plasmid (such as shown in Figure 1) and variants (such as Scpl, Scp2 and Scp3) from Saccharomyces cerevisiae (Volkert, et ah, 1989, Microbiological Reviews, 53, 299; Murray et al, 1988, J. MoI. Biol. 200, 601 ; Painting, et al, 1984, J. Applied Bacteriology, 56, 331). As a family of plasmids these molecules share a series of common features in that they typically possess two inverted repeats on opposite sides of the plasmid. have a similar size around 6-kbp (range 4757 to 6615-bp), three open reading frames, one of which encodes for a site specific recombinase (FLP) and an autonomously replicating sequence (ARS), also known as an origin of replication (orϊ), located close to the end of one of the inverted repeats. (Futcher, 1988, Yeast, 4, 27; Murray et ah, op. ciϊ., and Toh-e el at., 1986. Basic Life Sci. 40, 425). Despite their lack of discernible DNA sequence homology, their shared molecular architecture and the conservation of function of the three open reading frames have demonstrated a common ancestral link between the family members.
The above naturally occurring 2μm-family plasmids may, or may not, be used in the present invention, but this invention is not limited to the use of naturally occurring 2μm- family plasmids. For the purposes of this invention, a 2μm-family plasmid may, or may not, be as described below.
A 2μm-family plasmid is a circular, double stranded, DNA plasmid. It is typically small, such as between 3,000 to 10,000 bp. optionally between 4,500 to 7000 bp, excluding recombinantly inserted sequences.
A 2μm-family plasmid typically comprises at least three open reading frames ("ORFs") that each encodes a protein that functions in the stable maintenance of the 2μm-family plasmid as a multicopy plasmid. The proteins encoded by the three ORFs can be designated FLP, REPl and REP2. Where a 2μm-family plasmid comprises not all three of the ORFs encoding FLP, REPl and REP2 then ORFs encoding the missing protein(s) should be supplied in trans, either on another plasmid or by chromosomal integration.
A "FLP" protein is a protein capable of catalysing the site-specific recombination between inverted repeat sequences recognised by FLP. The inverted repeat sequences are termed FLP recombination target (FRT) sites and each is typically present as part of a larger inverted repeat (see below). Preferred FLP proteins comprise the sequence of the FLP proteins encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl , pPMl and the 2μm plasmid, for example as described in Volkert et al, op cit., Murray et al, op. cit., and Painting el al. , op. cit. Variants and fragments of these FLP proteins are also included in the present invention. "Fragments" and '"variants" are those which retain the ability of the native protein to catalyse the site-specific recombination between the same FRT sequences. Such variants and fragments will usually have at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with an FLP protein encoded by one of plasmids pSRl, pSBl, pSB2; pSB3, pSB4, pSML pKDl, pPMl and the 2μm plasmid. Different FLP proteins can have different FRT sequence specificities. A typical FRT site may, or may not, comprise a core nucleotide sequence flanked by inverted repeat sequences. In the 2μm plasmid. the FRT core sequence is 8 nucleotides in length and the flanking inverted repeat sequences are 13 nucleotides in length (Volkert et al, op. cit.). However the FRT site recognised by any given FLP protein may, or may not, be different to the 2μm plasmid FRT site.
REPl and REP2 are proteins involved in the partitioning of plasmid copies during cell division, and may, or may not, also have a role in the regulation of FLP expression. Considerable sequence divergence has been observed between REPl proteins from different 2μm-family plasmids, whereas no sequence alignment is possible between REP2 proteins derived from different 2μm-family plasmids. Preferred REPl and REP 2 proteins comprise the sequence of the REPl and REP 2 proteins encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2μm plasmid, for example as described in Volkert et al, op. cit. , Murray et al, op. cit. , and Painting et al, op. cit. Variants and fragments of these REPl and REP2 proteins are also included in the present invention. "Fragments" and "variants" of REPl and REP2 are those which, when encoded by the plasmid in place of the native ORP. do not substantially disrupt the stable multicopy maintenance of the plasmid within a suitable yeast population. Such variants and fragments of REPl and REP 2 will usually have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with a REPl and REP2 protein, respectively, as encoded by one of plasmids pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2μm plasmid.
The REPl and REP2 proteins encoded by the ORFs on the plasmid must be compatible. It is preferred that the REPl and REP2 proteins have the sequences of REPl and REP2 proteins encoded by the same naturally occurring 2μm-family plasmid, such as pSRl, pSBl, pSB2, pSB3, pSB4, pSMl, pKDl, pPMl and the 2μm plasmid, or variant or fragments thereof.
A 2μm-family plasmid typically comprises two inverted repeat sequences. The inverted repeats may be any size, so long as they each contain an FRT site (see above). The inverted repeats are typically highly homologous. They may, or may not, share greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%,
99.5% or more sequence identity. In a preferred embodiment they are identical.
Typically the inverted repeats are each between 200 to 1000 bp in length. Preferred inverted repeat sequences may, or may not, each have a length of from
200 to 300 bp, 300 to 400 bp, 400 to 500 bp, 500 to 600 bp, 600 to 700 bp, 700 to
800 bp, 800 to 900 bp, or 900 to 1000 bp. Particularly preferred inverted repeats are those of the plasmids pSRl (959 bp), pSBl (675 bp), pSB2 (477 bp), pSB3
(391 bp), pSMl (352 bp), pKDl (346 bp), the 2μm plasmid (599 bp), pSB4 or pPMl .
The sequences of the inverted repeats may, or may not, be varied. However, the sequences of the FRT site in each inverted repeat should be compatible with the specificity of the FLP protein encoded by the plasmid, thereby to enable the encoded FLP protein to act to catalyse the site-specific recombination between the inverted repeat sequences of the plasmid. Recombination between inverted repeat sequences (and thus the ability of the FLP protein to recognise the FRT sites with the plasmid) can be determined by methods known in the art. For example, a plasmid in a yeast cell under conditions that favour FLP expression can be assayed for changes in the restriction profile of the plasmid which would result from a change in the orientation of a region of the plasmid relative to another region of the plasmid. The detection of changes in restriction profile indicate that the FLP protein is able to recognise the FRT sites in the plasmid and therefore that the FRT site in each inverted repeat are compatible with the specificity of the FLP protein encoded by the plasmid.
In a particularly preferred embodiment, the sequences of inverted repeats, including the FRT sites, are derived from the same 2μm-family plasmid as the ORF encoding the FLP protein, such as pSRl, pSBl. pSB2, pSB3, pSB4, pSMl , pKDl. pPMl or the 2μm plasmid.
The inverted repeats are typically positioned with the 2μm-family plasmid such that the two regions defined between the inverted repeats (e.g. such as defined as UL and US in the 2μm plasmid) are of approximately similar size, excluding exogenously introduced sequences such as transgenes. For example, one of the two regions may, or may not, have a length equivalent to at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, up to 100%, of the length of the other region.
A 2μm-family plasmid typically comprises the ORF that encodes FLP and one inverted repeat (arbitrarily termed ''IRl" to distinguish it from the other inverted repeat mentioned in the next paragraph) juxtaposed in such a manner that IRl occurs at the distal end of the FLP ORF, without any intervening coding sequence, for example as seen in the 2μm plasmid. By '"distal end" in this context we mean the end of the FLP ORF opposite to the end from which the promoter initiates its transcription. In a preferred embodiment, the distal end of the FLP ORF overlaps with IRl .
A 2μm-family plasmid typically comprises the ORF that encodes REP2 and the other inverted repeat (arbitrarily termed "IR2" to distinguish it from IRl mentioned in the previous paragraph) juxtaposed in such a manner that IR2 occurs at the distal end of the REP 2 ORF, without any intervening coding sequence, for example as seen in the 2μm plasmid. By ''distal end" in this context we mean the end of the REP2 ORF opposite to the end from which the promoter initiates its transcription.
In one embodiment, the ORFs encoding REP2 and FLP may, or may not, be present on the same region of the two regions defined between the inverted repeats of the 2μm-family plasmid, which region may be the bigger or smaller of the regions (if there is any inequality in size between the two regions).
In one embodiment, the ORFs encoding REP2 and FLP may, or may not, be transcribed from divergent promoters.
Typically, the regions defined between the inverted repeats (e.g. such as defined as UL and US in the 2μm plasmid) of a 2μm-family plasmid may. or may not, comprise not more than two endogenous genes that encode a protein that functions in the stable maintenance of the 2μm-family plasmid as a multicopy plasmid. Thus in a preferred embodiment, one region of the plasmid defined between the inverted repeats may, or may not, comprise not more than the ORFs encoding FLP and REP 2; FLP and REPl; or REPl and REP 2, as endogenous coding sequence.
A 2μm-family plasmid typically comprises an origin of replication (also known as an ''autonomously replicating sequence - "ARS"), which is typically bidirectional. Any appropriate ARS sequence can be present. Consensus sequences typical of yeast chromosomal origins of replication may, or may not. be appropriate (Broach et άl, 1982; Cold Spring Harbor Symp. Quant. Biol, 47, 1165-1174; Williamson, Yeast, 1985, 1, 1-14). Preferred ARSs include those isolated from pSRL pSBl, pSB2, pSB3; pSB4, pSML pKDl, pPMl and the 2μm plasmid.
Thus, a preferred 2μm-family plasmid may. or may not. comprise ORFs encoding FLP, REPl and REP2, two inverted repeat sequences each inverted repeat comprising an FRT site compatible with the encoded FLP protein, and an ARS sequence. Preferably the FRT sites are derived from the same 2μm-family plasmid as the sequence of the encoded FLP protein. More preferably the sequences of the encoded REPl and REP2 proteins are derived from the same 2μm-family plasmid as each other. Even more preferably, the FRT sites are derived from the same 2μm-family plasmid as the sequence of the encoded FLP, REPl and REP2 proteins. Yet more preferably, the sequences of the ORFs encoding FLP, REPl and REP2, and the sequence of the inverted repeats (including the FRT sites) are derived from the same 2μm-family plasmid. Furthermore, the ARS site may, or may not, be derived from the same 2μm-family plasmid as one or more of the ORFs of FLP, REPl and REP 2, and the sequence of the inverted repeats (including the FRT sites).
The term "derived from" includes sequences having an identical sequence to the sequence from which they are derived. However, variants and fragments thereof, as defined above, are also included. For example, an FLP gene having a sequence derived from the FLP gene of the 2μm plasmid may, or may not, have a modified promoter or other regulator}' sequence compared to that of the naturally occurring gene. Additionally or alternatively, an FLP gene having a sequence derived from the FLP gene of the 2μm plasmid may, or may not, have a modified nucleotide sequence in the open reading frame which may, or may not, encode the same protein as the naturally occurring gene, or may, or may not, encode a modified FLP protein. The same considerations apply to other sequences on a 2μm-family plasmid having a sequence derived from a particular source.
Optionally, a 2μm-family plasmid may, or may not, comprise a region derived from the STB region (also known as REP3) of the 2μm plasmid, as defined in Volkert et al, op. cit. The STB region in a 2 μm -family plasmid of the invention may, or may not, comprise two or more tandem repeat sequences, such as three, four, five or more. Alternatively, no tandem repeat sequences may be present. The tandem repeats may be any size, such as 10, 20, 30, 40, 50, 60 70, 80, 90, 100 bp or more in length. The tandem repeats in the STB region of the 2μm plasmid are 62 bp in length. It is not essential for the sequences of the tandem repeats to be identical. Slight sequence variation can be tolerated. It may, or may not, be preferable to select an STB region from the same plasmid as either or both of the REPl and REP2 ORFs. The STB region is thought to be a cw-acting element and preferably is not transcribed.
Optionally, a 2μm-family plasmid may, or may not, comprise an additional ORF that encodes a protein that functions in the stable maintenance of the 2μm-family plasmid as a multicopy plasmid. The additional protein can be designated RAF or D. ORFs encoding the RAF or D gene can be seen on, for example, the 2μm plasmid and pSMl. Thus a RAF or D ORF can comprise a sequence suitable to encode the protein product of the RAF or D gene ORFs encoded by the 2μm plasmid or pSMl, or variants and fragments thereof. Thus variants and fragments of the protein products of the RAF or D genes of the 2μm plasmid or pSMl are also included in the present invention. "Fragments" and "variants" of the protein products of the RAF or D genes of the 2μm plasmid or pSMl are those which, when encoded by the 2μm plasmid or pSMl in place of the native ORF, do not disrupt the stable multicopy maintenance of the plasmid within a suitable yeast population. Such variants and fragments will usually have at least 5%, 10%, 20%, 30%, 40%, 50%, 60%; 70%, 80%, 90%, 95%, 98%, 99%, or more, homology with the protein product of the RAF or D gene ORFs encoded by the 2μm plasmid or pSMl .
A naturally occurring 2μm-family plasmid may, or may not. be preferred. A naturally occurring 2μm-family plasmid is any plasmid having the features defined above, which plasmid is found to naturally exist in yeast, i.e. has not been recombinantly modified to include heterologous sequence. Optionally the naturally occurring 2μm-family plasmid is selected from pSRl (Accession No. X02398), pSB3 (Accession No. X02608) or pSB4 as obtained from Zygosaccharomyces rouxii, pSBl or pSB2 (Accession No. NC_002055 or Ml 8274) both as obtained from Zygosaccharomyces bailli, pSMl (Accession No. NC_002054) as obtained from Zygosaccharomyces fermentati, pKDl (Accession No. X03961) as obtained from
Figure imgf000056_0001
drosophilarum, pPMl from Pichia membranaefaciens or, preferably, the 2μm plasmid (Accession No. NC_001398 or J01347) as obtained from Saccharomyces cerevisiae. Accession numbers in this paragraph refer to NCBI deposits.
The 2μm plasmid (Figure 1) is a 6,318-bp double-stranded DNA plasmid, endogenous in most Saccharomyces cerevisiae strains at 60-100 copies per haploid genome. The 2μm plasmid comprises a small unique (US) region and a large unique (UL) region, separated by two 599-bp inverted repeat sequences. Site-specific recombination of the inverted repeat sequences results in inter- conversion between the A-form and B-form of the plasmid in vivo (Volkert & Broach, 1986, Cell, 46, 541). The two forms of 2μm differ only in the relative orientation of their unique regions.
While DNA sequencing of a cloned 2μm plasmid (also known as Scpl) from
Saccharomyces cerevisiae gave a size of 6,318-bp (Hartley and Donelson, 1980,
Nature, 286, 860), other slightly smaller variants of 2μm, Scp2 and Scp3, are known to exist as a result of small deletions of 125-bp and 220-bp, respectively, in a region known as STB (Cameron el al, 1977. Nucl Acids Res., 4. 1429: Kikuchi, 1983, Cell 35, 487 and Livingston & Hahne, 1979, Proc. Natl. Acad Sci USA, 16, 3727). In one study about 80% of natural Saccharomyces strains from around the world contained DNA homologous to 2μm (by Southern blot analysis) (HoIl enberg, 1982, Current Topics in Microbiology/ and Immunobiology, 96, 119). Furthermore, λ'ariation (genetic polymorphism) occurs within the natural population of 2μm plasmids found in S. cerevisiae and S. carlsbergensis, with the NCBI sequence (accession number NC_001398) being one example.
The 2μm plasmid has a nuclear localisation and displays a high level of mitotic stability (Mead et al, 1986, Molecular & General Genetics, 205, 417). The inherent stability of the 2μm plasmid results from a plasmid-encoded copy number amplification and partitioning mechanism, which can be compromised during the development of chimeric vectors (Futcher & Cox. 1984, J. Bacleriol, 157, 283; Bachmair & Ruis, 1984, Monatshefte fur Chemie, 115, 1229). A yeast strain, which contains a 2μm plasmid is known as [cir+], while a yeast strain which does not contain a 2μm plasmid is known as [cir0].
The US-region of the 2μm plasmid contains the REP2 and FLP genes, and the UL-region contains the REPl and D (also known as RAF) genes, the STB-locus and the origin of replication (Broach & Hicks, 1980, Cell, 21, 501; Sutton &
Broach, 1985, MoI. Cell. Biol, 5, 2770). The FIp recombinase binds to FRT-sites
(FIp Recognition Target) within the inverted repeats to mediate site-specific recombination, which is essential for natural plasmid amplification and control of plasmid copy number in vivo (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A.,
82, 7270; Jayaram, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 5875). The copy number of 2μm-family plasmids can be significantly affected by changes in FIp recombinase activity (Sleep et al, 2001, Yeast, 18, 403; Rose & Broach, 1990,
Methods Enzymol , 185, 234). The Repl and Rep2 proteins mediate plasmid segregation, although their mode of action is unclear (Sengupta et al, 2001, J. Bacteriol, 183, 2306). They also repress transcription of the FLP gene (Reynolds et al, 1987, Mo/. Cell Biol, 7, 3566).
The FLP and REP2 genes of the 2μm plasmid are transcribed from divergent promoters, with apparently no intervening sequence defined between them. The FLP and REP2 transcripts both terminate at the same sequence motifs within the inverted repeat sequences, at 24-bp and 178-bp respectively after their translation termination codons (Sutton & Broach, 1985, MoJ. Cell. Biol, S, 2770).
In the case of FLP, the C-terminal coding sequence also lies within the inverted repeat sequence. Furthermore, the two inverted repeat sequences are highly conserved over 599-bp, a feature considered advantageous to efficient plasmid replication and amplification in vivo, although only the FRT-sites (less than 65- bp) are essential for site-specific recombination in vitro (Senecoff et al, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 7270; Jayaram, 1985, Proc. Natl. Acad. Sci. U.S.A., 82, 5875; Meyer-Leon et al, 1984, Cold Spring Harbor Symposia On Quantitative Biolog}', 49, 797). The key catalytic residues of FIp are arginine-308 and tyrosine-343 (which is essential) with strand-cutting facilitated by histidine- 309 and histidine 345 (Prasad et al, 1987, Proc. Natl Acad. Sci. U.S.A., 84, 2189; Chen et al, 1992, Cell, 69, 647; Grainge et al, 2001, J. MoI. Biol, 314, 717).
Two functional domains are described in Rep2. Residues 15-58 form a Repl- binding domain, and residues 59-296 contain a self-association and STB-binding region (Sengupta et al, 2001, J. Bacteriol, 183, 2306).
Chimeric or large deletion mutant derivatives of 2μm which lack many of the essential functional regions of the 2μm plasmid but retain the functional cis element ARS and STB, cannot effectively partition between mother and daughter cells at cell division. Such plasmids can do so if these functions are supplied in trans, by for instance the provision of a functional 2μm plasmid within the host, such as a [cir+] host.
Genes of interest have previously been inserted into the UL-region of the 2μm plasmid. For example, see plasmid pSACSUl in EP 0 286 424 and the plasmid shown in Figure 2 of WO 2005/061718, which includes a β-lactamase gene (for ampicillin resistance), a LEU2 selectable marker and an oligonucleotide linker, the latter two of which are inserted into a unique SnaBl-ήXt within the UL-region of the 2μm-like disintegration vector, pSAC3 (see EP 0 286 424). The E. coli DNA between the A^αl-sites that contains the ampicillin resistance gene is lost from the plasmid shown in Figure 2 of WO 2005/061718 after transformation into yeast. This is described in Chinery & Hinchliffe, 1989, Curr. Genet., 16, 21 and EP 0 286 424, where these types of vectors are designated "disintegration vectors". Further polynucleotide insertions can be made in a Tvorf-site within a linker (Sleep et al, 1991, Biotechnology (N Y), 9, 183).
Alternative insertion sites in 2μm plasmid are known in the art, including those described in Rose & Broach (1990, Methods Enzymol, 185, 234-279), such as plasmids pCV19, pCV20, CVneo, which utilise an insertion at FcoRI in FZP, plasmids pCV21, pGT41 and pYE which utilise EcoRl in D as the insertion site, plasmid pHKB52 which utilises Pstl in D as the insertion site, plasmid pJDB248 which utilises an insertion at Pstl in D and EcoRl in D, plasmid p.TDB219 in which Pstl in D and EcoRl in FLP are used as insertion sites, plasmid Gl 8, plasmid pAB18 which utilises an insertion at CIcA in FiP, plasmids pGT39 and pA3, plasmids pYTl l, pYT14 and pYTl l-LEU which use Pstl in D as the insertion site, and plasmid PTY39 which uses FcoRI in FLP as the insertion site. Other 2μm plasmids include pSAC3, ρSAC3Ul, pSAC3U2, pSAC300, pSAC310, pSAC3Cl, pSAC3PLl, pSAC3SL4, and pSAC3SCl are described in EP 0 286 424 and Chinery & Hinchliffe (1989, Curr. Genet, 16, 21-25) which also described Pstl, Eagl or SnctBl as appropriate 2μm insertion sites. Further 2μm plasmids include pAYE255. pAYE316, pAYE443, pAYE522 (Kerry-Williams et al, 1998, Yeast, 14, 161 -169), pDB2244 (WO 00/44772), and pAYE329 (Sleep et al, 2001, Yeast, 18, 403-421).
In one preferred embodiment, one or more genes are inserted into a 2μm-family plasmid within an untranscribed region around the ARS sequence. For example, in the 2μm plasmid obtained from S. cerevisiae, the untranscribed region around the ARS sequence extends from the end of the D gene to the beginning of ARS sequence. Insertion into SnaBI (near the origin of replication sequence ARS) is described in Chinery & Hinchliffe, 1989, Curr. Genet., 16, 21-25. The skilled person will appreciate that gene insertions can also be made in the untranscribed region at neighbouring positions to the SnaBI site described in Chinery & Hinchliffe.
In another preferred embodiment, REP2 and FLP genes in a 2μm-family plasmid each have an inverted repeat adjacent to them, and one or more genes are inserted into a 2μm-family plasmid within the region between the first base after the last functional codon of either the REP2 gene or the FLP gene and the last base before the FRT site in the inverted repeat adjacent to said gene. The last functional codon of either a REP2 gene or a FLP gene is the codon in the open reading frame of the gene that is furthest downstream from the promoter of the gene whose replacement by a stop codon will lead to an unacceptable loss of multicopy stability of the plasmid, as defined herein. Thus, disruption of the REP 2 or FLP genes at any point downstream of the last functional codon in either gene, by insertion of a polynucleotide sequence insertion, deletion or substitution will not lead to an unacceptable loss of multicopy stability of the plasmid.
For example, the REP2 gene of the 2μm plasmid can be disrupted after codon 59 and that the FLP gene of the 2μm plasmid can be disrupted after codon 344, each without a loss of multicopy stability of the plasmid. The last functional codon in equivalent genes in other 2μm-family piasmids can be determined routinely by making mutants of the piasmids in either the FLP or REP 2 genes and following the tests set out herein to determine whether the plasmid retains multicopy stability. Thus, a plasmid insertion site as defined in WO 2005/061719 may, or may not, be used to carry one or more a recombinant genes according to any aspect of the present invention.
One can determine whether a plasmid retains multicopy stability using test such as defined in Chinery & Hinchliffe (1989, Curr. Genet., 16, 21-25). For yeast that do not grow in the non-selective media (YPD, also designated YEPD) defined in Chinery & Hinchliffe (1989, Curr. Genet., 16, 21-25) other appropriate nonselective media might be used. Plasmid stability may be defined as the percentage cells remaining prototrophic for the selectable marker after a defined number of generations. The number of generations will preferably be sufficient to show a difference between a control plasmid. such as pSAC35 or pSACSIO, or to shown comparable stability to such a control plasmid. The number of generations may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more. Higher numbers are preferred. The acceptable plasmid stability might be 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or substantially 100%. Higher percentages are preferred. The skilled person will appreciate that, even though a plasmid may have a stability less than 100% when grown on non-selective media, that plasmid can still be of use when cultured in selective media. For example plasmid pDB2711 as described in the examples is only 10% stable when the stability is determined accordingly to test of Example 2 of WO 2005/061719, but provides a 15-fold increase in recombinant transferrin productivity in shake flask culture under selective growth conditions.
Thus one or more gene insertions may. or may not, occur between the first base after the last functional codon of the REP2 gene and the last base before the FRT site in an inverted repeat adjacent to said gene, preferably between the first base of the inverted repeat and the last base before the FRT site, more preferably at a position after the translation termination codon of the REP2 gene and before the last base before the FRT site.
Additionally or alternatively one or more gene insertions may, or may not. occur between the first base after the last functional codon of the FLP gene and the last base before the FRT site in an inverted repeat adjacent to said gene, preferably between the first base of the inverted repeat and the last base before the FRT site, more preferably between the first base after the end of the FLP coding sequence and the last base before the FRT site, such as at the first base after the end of the FLP coding sequence.
In one preferred embodiment, where the 2μm-family plasmid is based on the 2μm plasmid of S cerevisiae, it is a disintegration vector as known in the art (for example, see EP 286 424. the contents of which are incorporated herein by reference). A disintegration vector may, or may not, be a 2μm plasmid vector comprising a DNA sequence which is intended to be lost by recombination, three
2μm FRT sites, of which one pair of sites is in direct orientation and the other two pairs are in indirect orientation, and a DNA sequence of interest (such as an E. coli origin of replication and bacterial selectable marker), the said sequence to be lost being located between the said sites which are in direct orientation.
Thus, the sequence to be lost may, or may not, comprise a selectable marker DNA sequence.
A preferred disintegration vector may, or may not, comprise a complete 2μm plasmid additionally carrying (i) a bacterial plasmid DNA sequence necessary for propagation of the λ'ector in a bacterial host; (ii) an extra 2μm FRT site; and a selectable marker DNA sequence for yeast transformation; the said bacterial plasmid DNA sequence being present and the extra FRT site being created at a restriction site, such as Xbal, in one of the two inverted repeat sequences of the 2μm plasmid. the said extra FRT site being in direct orientation in relation to the endogenous FRT site of the said one repeat sequence, and the bacterial plasmid DNA sequence being sandwiched between the extra FRT site and the endogenous FRT site of the said one repeat sequence. In a preferred disintegration vector, all bacterial plasmid DNA sequences may, or may not, be sandwiched as said. A particularly preferred 2μm plasmid vector has substantially the configuration of pSAC3 as shown in EP 286 424.
The term "disintegration vector" as used herein also includes plasmids as defined in US 6,451,559. the contents of which are incorporated herein by reference. Thus a disintegration vector may, or may not, be a 2μm vector that, other than DNA sequence encoding non-yeast polypeptides, contains no bacterial (particularly E. coif) origin of replication, or more preferably no bacterial (particularly E. coli) sequence and preferably all DNA in said vector, other than DNA sequence encoding non-yeast polypeptides, is yeast-derived DNA.
Desired proteins and other proteins defined by the present application:
The terms "protein" and "desired protein" as used herein includes all natural and non-natural proteins, polypeptides and peptides. For the purposes of the present invention, a "heterologous protein" is a protein that is encoded by a "recombinant gene" as described above. The "heterologous protein" may, or may not, be identical in sequence to a protein that is encoded by one of more other genes that naturally occur in the expression system that is used (by "expression system" we include the meaning of a host cell's genome (typically the chromosome) where the "recombinant gene" is chromosomally integrated, or a plasmid where the "recombinant gene" is encoded by a plasmid). For example, in the context of a "heterologous protein" that is encoded by a "recombinant gene" carried on a 2μm- family plasmid, the "heterologous protein'" may. or may not. be a protein that is not naturally encoded by a 2μm-family plasmid and can also be described as a "non 2μm-family plasmid protein". For convenience, the terms "heterologous protein" and "non 2μm-family plasmid protein" are used synonymously in this application. Optionally therefore, when encoded by a 2μm-family, the heterologous protein is not a FLP, REPl, REP 2, or a RAF/D protein as encoded by any one of pSRl, pSB3 or pSB4 as obtained from Z. rouxii, pSBl or pSB2 both as obtained from Z. bailli, pSMl as obtained from Z fermentati, pKDl as obtained from K. drosophilarum, pPMl as obtained from P. membranaefaciens or the 2μm plasmid as obtained from S. cerevisiae.
A gene encoding a desired heterologous, or other, protein comprises a polynucleotide sequence encoding the heterologous protein (typically according to standard codon usage for any given organism), designated the open reading frame ("ORF"). The gene may, or may not, additionally comprise some polynucleotide sequence that does not encode an open reading frame (termed "non-coding region").
Non-coding region in the gene may, or may not, contain one or more regulatory sequences, operatively linked to the ORF, which allow for the transcription of the open reading frame and/or translation of the resultant transcript.
The term "regulatory sequence" refers to a sequence that modulates (i.e., promotes or reduces) the expression (i.e., the transcription and/or translation) of an ORF to which it is operably linked. Regulatory regions typically include promoters, terminators, ribosome binding sites and the like. The skilled person will appreciate that the choice of regulatory region will depend upon the intended expression system. For example, promoters may, or may not, be constitutive or inducible and may, or may not. be cell- or tissue -type specific or non-specific. Suitable regulatory regions, may, or may not. be 5bp. lObp, 15bp. 20bp, 25bp. 30bp; 35bp, 40bp; 45bρ, 50bp, 60bp: 70bp, 80bp, 90bp, lOObp, 120bp; 140bp; 160bp, 180bp, 200bp, 220bp: 240bp, 260bp. 280bp, 300bp, 35Obp, 400bp, 450bp, 500bp, 550bp, 600bp, 650bp, 700bp, 750bp, 800bp; 85Obp, 900bp; 950bp, lOOObp, 1 lOObp, 1200bρ, 1300bp, 1400bp, 1500bp or greater, in length.
Those skilled in the art will recognise that the gene encoding a chaperone, for example PDI, may, or may not, additionally comprise non-coding regions and/or regulatory regions. Such non-coding regions and regulatory regions are not restricted to the native non-coding regions and/or regulatory regions normally associated with the chaperone ORP.
Where the expression system is yeast, such as Saccharomyces cerevisiae, suitable promoters for S. cerevisiae include those associated with the PGKl gene, GALl or GALlO genes, TEFl1 TEF2, PYKl, PMAl, CYCl, PHO5, TRPl, ADHl, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase. α-mating factor pheromone, a-mating factor pheromone, the PRBl promoter, the PRAl promoter, the GPDl promoter, and hybrid promoters involving hybrids of parts of 5' regulator}' regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067).
Suitable transcription termination signals are well known in the art. Where the host cell is eucaryotic, the transcription termination signal is optionally derived from the 3' flanking sequence of a eucaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, or may not, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may, or may not, correspond to the promoter. Alternatively, they may be different. In that case, and where the host is a yeast, optionally S. cerevisiae. then the termination signal of the S. cerevisiae ADHl, ADH2, CYCL, or PGKl genes are preferred.
It may, or may not. be beneficial for the promoter and open reading frame of the gene, such as a gene encoding the chaperone (e.g. PDIl) or a desired protein (such as a heterologous desired protein), to be flanked by transcription termination sequences so that the transcription termination sequences are located both upstream and downstream of the promoter and open reading frame, in order to prevent transcriptional read-through into neighbouring genes, such as 2μm genes, and vice versa.
In one embodiment, the favoured regulatory sequences in yeast, such as Saccharomyces cerevisiae, include: a yeast promoter (e.g. the Saccharomyces cerevisiae PRBl promoter), as taught in EP 431 880; and a transcription terminator, optionally the terminator from Saccharomyces ADHl, as taught in EP 60 057. Optionally, the vector incorporates at least two translation stop codons.
It may, or may not, be beneficial for the non-coding region to incorporate more than one DNA sequence encoding a translational stop codon, such as UAA, UAG or UGA, in order to minimise translational read-through and thus avoid the production of elongated, non-natural fusion proteins. The translation stop codon UAA is preferred.
The term "operably linked" includes within its meaning that a regulatory sequence is positioned within any non-coding region in a gene such that it forms a relationship with an ORF that permits the regulatory region to exert an effect on the ORP in its intended manner. Thus a regulatory region "operably linked" to an
ORF is positioned in such a way that the regulator}' region is able to influence transcription and/or translation of the ORF in the intended manner, under conditions compatible with the regulatory sequence. In one preferred embodiment, the desired protein (such as the heterologous desired protein) is secreted. In that case, a sequence encoding a secretion leader sequence which, for example, comprises most of the natural HSA secretion leader, plus a small portion of the S cerevisiae α-mating factor secretion leader as taught in WO 90/01063 may, or may not, be included in the open reading frame.
Alternatively, the desired protein (such as a heterologous desired protein) may, or may not, be intracellular.
The desired protein (such as a heterologous desired protein) may, or may not, comprise the sequence of a eucaryotic protein, or a fragment or variant thereof. Suitable eucaryotes include fungi, plants and animals. In one embodiment the heterologous protein may, or may not, be a fungal protein, such as a yeast protein. In another preferred embodiment the desired protein (such as a heterologous desired protein) may, or may not, be an animal protein. Exemplary animals include vertebrates and invertebrates. Exemplar}' vertebrates include mammals, such as humans, and non-human mammals.
Thus the desired protein (such as a heterologous desired protein) may, or may not, comprise the sequence of a yeast protein. It may. or may not, for example, comprise the sequence of a yeast protein from the same host from which a 2μm- family plasmid is derived, particularly if the gene encoding the heterologous protein is integrated into said 2μm-family plasmid. Those skilled in the art will recognise that a method, use or plasmid of the invention may, or may not, comprise DNA sequences encoding more than one heterologous protein, more than one chaperone, or more than one heterologous protein and more than one chaperone. In another embodiment, the desired protein (such as a desired heterologous protein) may, or may not, comprise the sequence of albumin, a monoclonal antibody, an etoposide. a serum protein (such as a blood clotting factor), antistasin, a tick anticoagulant peptide, transferrin, lactoferrin. endostatin. angiostatin. collagens, immunoglobulins or immunoglobulin-based molecules or fragment of either (e.g. a Small Modular ImmunoPharmaceutical™ ("'SMIP") or dAb, Fab' fragments, F(ab')2, scAb, scFv or scFv fragment), a Kunitz domain protein (such as those described in WO 03/066824, with or without albumin fusions), interferons, interleukins, ILlO, ILI l, IL2, interferon α species and sub- species, interferon β species and sub-species, interferon γ species and sub-species, leptin, CNTF, CNTF Axis, ILl -receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5- helix, T20 peptide, Tl 249 peptide, HIV gp41, HIV gpl20, urokinase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-like peptides, insulin-like growth factor, calcitonin, growth hormone, transforming growth factor β, tumour necrosis factor, G-CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and active forms, including but not limited to plasminogen, fibrinogen, thrombin, pre-thrombin. prothrombin, von Willebrand's factor, o.\ -antitrypsin, plasminogen activators, Factor VII, Factor VIII, Factor IX, Factor X and Factor XIII, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, or a variant or fragment of any of the above.
A "variant", in the context of the above-listed proteins, refers to a protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example enzymatic activity or receptor binding (type of and specific activity), thermostability, activity in a certain pH-range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
By "conservative substitutions" is intended combinations such as VaI, lie. Leu. Ala. Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy, Ala; Lys, Arg, His; and Phe. Tyr, Trp. Preferred conservative substitutions include GIy, Ala; VaI, He, Leu; Asp. GIu: Asn. GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
A '-variant" typically has at least 25%, at least 50%, at least 60% or at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, yet more preferably at least 99%, most preferably at least 99.5% sequence identity to the polypeptide from which it is derived.
The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally.
The alignment may alternatively be carried out using the Clustal W program (Thompson et al, (1994) Nucleic Acids Res., 22(22), 4673-80). The parameters used may, or may not, be as follows:
• Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.
• Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.
• Scoring matrix: BLOSUM. Such variants may. or may not, be natural or made using the methods of protein engineering and site-directed mutagenesis as are well known in the art.
A "fragment", in the context of the above-listed proteins, refers to a protein wherein at one or more positions there have been deletions. Thus the fragment may, or may not. comprise at most 5. 10. 20, 30, 40 or 50% of the complete sequence of the full mature polypeptide. Typically a fragment comprises up to 60%, more typically up to
70%, preferably up to 80%. more preferably up to 90%. even more preferably up to
95%, yet more preferably up to 99% of the complete sequence of the full desired protein. Particularly preferred fragments of a protein comprise one or more whole domains of the protein.
In one particularly preferred embodiment the desired protein (such as a desired heterologous protein) comprises the sequence of albumin or a variant or fragment thereof.
By "albumin" we include a protein comprising the sequence of an albumin protein obtained from any source. Typically the source is mammalian. In one preferred embodiment the serum albumin is human serum albumin ("HSA"). The term "human serum albumin'" includes the meaning of a serum albumin having an amino acid sequence naturally occurring in humans, and variants thereof. Optionally the albumin has the amino acid sequence disclosed in WO 90/13653 or a variant thereof. The HSA coding sequence is obtainable by known methods for isolating cDNA corresponding to human genes, and is also disclosed in, for example, EP 73 646 and EP 286 424.
In another preferred embodiment the "albumin" comprises the sequence of bovine serum albumin. The term "bovine serum albumin" includes the meaning of a serum albumin having an amino acid sequence naturally occurring in cows, for example as taken from Swissprot accession number P02769, and variants thereof as defined below. The term "bovine serum albumin'" also includes the meaning of fragments of full-length bovine serum albumin or variants thereof, as defined below.
In another preferred embodiment the albumin comprises the sequence of an albumin derived from one of serum albumin from dog (e.g. see Swissprot accession number P49822), pig (e.g. see Swissprot accession number P08835), goat (e.g. as available from Sigma as product no. A2514 or A4164), turkey (e.g. see Swissprot accession number 073860), baboon (e.g. as available from Sigma as product no. Al 516), cat (e.g. see Swissprot accession number P49064), chicken (e.g. see Swissprot accession number P19121), ovalbumin (e.g. chicken ovalbumin) (e.g. see Swissprot accession number POl 012), donkey (e.g. see Swissprot accession number P39090), guinea pig (e.g. as available from Sigma as product no. A3060, A2639, 05483 or A6539), hamster (e.g. as available from Sigma as product no. A5409), horse (e.g. see Swissprot accession number P35747), rhesus monkey (e.g. see Swissprot accession number Q28522), mouse (e.g. see Swissprot accession number O89020). pigeon (e.g. as defined by Khan et al, 2002, Int. J. Biol. Macromol, 30(3-4),171-8), rabbit (e.g. see Swissprot accession number P49065), rat (e.g. see Swissprot accession number P36953) and sheep (e.g. see Swissprot accession number P14639) and includes variants and fragments thereof as defined below.
Many naturally occurring mutant forms of albumin are known. Many are described in Peters. (1996, All About Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, Inc., San Diego, California, p.170-181). A variant as defined above may, or may not, be one of these naturally occurring mutants.
A "variant albumin" refers to an albumin protein wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in an albumin protein for which at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolality (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH- stability) has not significantly been changed. "'Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.
By '"conservative substitutions" is intended combinations such as GIy, Ala; VaI, He, Leu; Asp, GIu: Asn, GIn; Ser, Thr; Lys. Arg; and Phe, Tyr. Such variants may, or may not, be made by techniques well known in the art, such as by site-directed mutagenesis as disclosed in US Patent No 4,302,386 issued 24 November 1981 to Stevens, incorporated herein by reference.
Typically an albumin variant will have more than 40%, usually at least 50%, more typically at least 60%, preferably at least 70%, more preferably at least 80%, yet more preferably at least 90%, even more preferably at least 95%, most preferably at least 98% or more sequence identity with naturally occurring albumin. The percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson el ah, 1994). The parameters used may, or may not, be as follows:
Fast pairwise alignment parameters: K-tuple(word) size; I5 window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.
The term "fragment" as used above includes any fragment of full-length albumin or a variant thereof, so long as at least one basic property, for example binding activity (type of and specific activity e.g. binding to bilirubin), osmolarity (oncotic pressure, colloid osmotic pressure), behaviour in a certain pH-range (pH-stability) has not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein. A fragment wall typically be at least 50 amino acids long. A fragment may, or may not, comprise at least one whole sub-domain of albumin. Domains of HSA have been expressed as recombinant proteins (Dockal, M. et al, 1999, J. Biol. Chem., 274, 29303- 29310), where domain I was defined as consisting of amino acids 1-197, domain II was defined as consisting of amino acids 189-385 and domain III was defined as consisting of amino acids 381-585. Partial overlap of the domains occurs because of the extended α-helix structure (hlθ-hl ) which exists between domains I and II, and between domains II and III (Peters, 1996, op. cit., Table 2-4). HSA also comprises six sub-domains (sub-domains IA, IB, HA, HB, IHA and IHB). Sub-domain IA comprises amino acids 6-105, sub-domain IB comprises amino acids 120-177, sub-domain HA comprises amino acids 200-291, sub-domain HB comprises amino acids 316-369, sub-domain IIIA comprises amino acids 392-491 and sub-domain HIB comprises amino acids 512-583. A fragment may, or may not, comprise a whole or part of one or more domains or sub-domains as defined above, or any combination of those domains and/or sub-domains.
In another particularly preferred embodiment the desired protein (such as a desired heterologous protein) comprises the sequence of transferrin or a variant or fragment thereof. The term "transferrin" as used herein includes all members of the transferrin family (Testa, Proteins of iron metabolism, CRC Press, 2002; Harris & Aisen, Iron carriers and iron proteins, Vol. 5, Physical Bioinorganic Chemistry, VCH, 1991) and their derivatives, such as transferrin, mutant transferrins (Mason et al, 1993, Biochemistry, 32, 5472; Mason et al, 1998, Biochem. J, 330(1), 35), truncated transferrins, transferrin lobes (Mason et al, 1996, Protein Expr. PuHf. t 8, 1 19; Mason et al, 1991, Protein Expr. PuHf. t 2, 214), lactoferrin, mutant lactoferrins, truncated lactoferrins, lactoferrin lobes or fusions of any of the above to other peptides, polypeptides or proteins (Shin et al, 1995, Proc. Natl. Acad. Sci. USA, 92, 2820; AIi et al, 1999, J. Biol. Chem., 274, 24066; Mason et al, 2002, Biochemistry, 4I5 9448).
The transferrin may, or may not, be human transferrin. The term "human transferrin" is used herein to denote material which is indistinguishable from transferrin derived from a human or which is a variant or fragment thereof. A "variant" includes insertions, deletions and substitutions, either conservative or non-conservative, where such changes do not substantially alter the useful ligand- binding or immunogenic properties of transferrin.
Mutants of transferrin are included in the invention. Such mutants may, or may not, have altered immunogenicity. For example, transferrin mutants may, or may not, display modified (e.g. reduced) glycosylation. The N-linked glycosylation pattern of a transferrin molecule can be modified by adding/removing amino acid glycosylation consensus sequences such as N-X-S/T, at any or all of the N, X, or S/T position. Transferrin mutants may, or may not, be altered in their natural binding to metal ions and/or other proteins, such as transferrin receptor. An example of a transferrin mutant modified in this manner is exemplified below.
We also include naturally-occurring polymorphic variants of human transferrin or human transferrin analogues. Generally, variants or fragments of human transferrin will have at least 5%, 10%, 15%, 20%, 30%, 40% or 50% (preferably at least 80%, 90% or 95%) of human transferrin's ligand binding activity (for example iron-binding), weight for weight. The iron binding activity of transferrin or a test sample can be determined spectrophotometrically by 470nm:280nm absorbance ratios for the proteins in their iron-free and fully iron-loaded states. Reagents should be iron-free unless stated otherwise. Iron can be removed from transferrin or the test sample by dialysis against 0.1M citrate, 0.1 M acetate, 1OmM EDTA pH4.5. Protein should be at approximately 20mg/mL in 10OmM HEPES, 1OmM NaHCO3 pH8.0. Measure the 470nm:280nm absorbance ratio of apo- transferrin (Calbiochem, CN Biosciences, Nottingham, UK) diluted in water so that absorbance at 280nm can be accurately determined spectrophotometrically (0% iron binding). Prepare 2OmM iron-nitrilotriacetate (FeNTA) solution by dissolving 191mg nitrotriacetic acid in 2mL IM NaOH, then add 2mL 0.5M ferric chloride. Dilute to 5OmL with deionised water. Fully load apo-transferrin with iron (100% iron binding) by adding a sufficient excess of freshly prepared 2OmM FeNTA, then dialyse the holo-transferrin preparation completely against 10OmM HEPES, 1OmM NaHCO3 pH8.0 to remove remaining FeNTA before measuring the absorbance ratio at 470nm:280nm. Repeat the procedure using test sample, which should initially be free from iron, and compare final ratios to the control.
Additionally, single or multiple heterologous fusions comprising any of the above; or single or multiple heterologous fusions to albumin, transferrin or immunoglobins or a variant or fragment of any of these may, or may not. be used. Such fusions include albumin N-terminal fusions, albumin C-terminal fusions and co-N-terminal and C-terminal albumin fusions as exemplified by WO 01/79271, and transferrin N-terminal fusions, transferrin C-terminal fusions, and co-N- terminal and C-terminal transferrin fusions.
Examples of transferrin fusions are given in US patent applications US2003/0221201 and US2003/0226155, Shin, et al., 1995, Proc Natl Acad Sci USA, 92, 2820, AIi, et al, 1999, J Biol Chem, 214, 24066, Mason, et al., 2002, Biochemistry, 41, 9448. the contents of which are incorporated herein by reference.
The skilled person will also appreciate that the open reading frame of any other gene or variant, or part or either, can be utilised as an open reading frame for use with the present invention. For example, the open reading frame may, or may not, encode a protein comprising any sequence, be it a natural protein (including a zymogen), or a variant, or a fragment (which may, or may not, for example, be a domain) of a natural protein; or a totally synthetic protein; or a single or multiple fusion of different proteins (natural or synthetic). Such proteins can be taken, but not exclusively, from the lists provided in WO 01/79258, WO 01/79271, WO 01/79442, WO 01/79443, WO 01/79444 and WO 01/79480, or a variant or fragment thereof; the disclosures of which are incorporated herein by reference. Although these patent applications present the list of proteins in the context of fusion partners for albumin, the present invention is not so limited and, for the purposes of the present invention, any of the proteins listed therein may, or may not, be presented alone or as fusion partners for albumin, the Fc region of immunoglobulin, transferrin, lactoferrin or an)' other protein or fragment or variant of any of the above, as a desired polypeptide.
The desired protein (such as a desired heterologous protein) may, or may not, be a therapeutically active protein. In other words, it may, or may not, have a recognised medical effect on individuals, such as humans. Many different types of therapeutically active protein are well known in the art.
The desired protein (such as a desired heterologous protein) may, or may not, be a protein that is useful in diagnostic techniques. Many different types of diagnostically useful protein are well known in the art.
The desired protein (such as a desired heterologous protein) may, or may not, be a protein that has no relationship to healthcare. It may, or may not, for example, be a protein that has a utility as an industrial, domestic or nutritional (e.g. as a foodstuff or additive) agent. Many different types of proteins having industrial, domestic and/or nutritional utilities are also well known in the art.
The desired protein (such as a desired heterologous protein) may, or may not, comprise a leader sequence effective to cause secretion in a host cell, such as in a yeast cell.
Numerous natural or artificial polypeptide signal sequences (also called secretion pre regions) have been used or developed for secreting proteins from host cells. The signal sequence directs the nascent protein towards the machinery of the cell that exports proteins from the cell into the surrounding medium or. in some cases, into the periplasmic space. The signal sequence is usually, although not necessarily, located at the N-terminus of the primary translation product and is generally, although not necessarily, cleaved off the protein during the secretion process, to yield the '"mature" protein.
In the case of some proteins the entity that is initially secreted, after the removal of the signal sequence, includes additional amino acids at its N-terminus called a "pro" sequence, the intermediate entity being called a "pro-protein". These pro sequences may, or may not, assist the final protein to fold and become functional, and are usually then cleaved off. In other instances, the pro region simply provides a cleavage site for an enzyme to cleave off the pre-pro region and is not known to have another function.
The pro sequence can be removed either during the secretion of the protein from the cell or after export from the cell into the surrounding medium or periplasmic space.
Polypeptide sequences which direct the secretion of proteins, whether they resemble signal (i.e. pre) sequences or pre-pro secretion sequences, are referred to as leader sequences. The secretion of proteins is a dynamic process involving translation, translocation and post-translational processing, and one or more of these steps may not necessarily be completed before another is either initiated or completed.
For production of proteins in eucaryotic species such as the yeasts Saccharomyces cerevisiae, Zygosaccharomyces species,
Figure imgf000077_0001
lactis and Pichia past oris, known leader sequences include those from the S. cerevisiae acid phosphatase protein (Pho5p) (see EP 366 400), the invertase protein (Suc2p) (see Smith et al.
(1985) Science, 229, 1219-1224) and heat-shock protein-150 (Hspl50p) (see WO 95/33833). Additionally, leader sequences from the S. cerevisiae mating factor alpha- 1 protein (MFa- 1) and from the human lysozyme and human serum albumin (HSA) protein have been used, the latter having been used especially, although not exclusively, for secreting human albumin. WO 90/01063 discloses a fusion of the MFa- 1 and HSA leader sequences, which advantageously reduces the production of a contaminating fragment of human albumin relative to the use of the MFa- 1 leader sequence. Modified leader sequences are also disclosed in WO 2004/009819 and in the examples of this application; the reader will appreciate that those leader sequences can be used with proteins other than transferrin. In addition, the natural transferrin leader sequence may, or may not, be used to direct secretion of transferrin and other heterologous proteins.
Where a chaperone that is recombinantly expressed according to the present invention is protein disulphide isomerase, then optionally the desired protein (such as a desired heterologous protein) may, or may not, comprise disulphide bonds in its mature form. Any disulphide bonds may, or may not, be intramolecular and/or intermolecular.
The desired protein (such as a desired heterologous protein) may. or may not, be a commercially useful protein, such as a therapeutically, diagnostically, industrially, domestically or nutritionally useful protein. Some proteins, such as heterologously expressed proteins, are intended to interact with the cell in which they are expressed in order to bring about a beneficial effect on the cell's activities. These proteins are not, in their own right, commercially useful. Commercially useful proteins are proteins that have a utility ex vivo of the cell in which they are expressed. Nevertheless, the skilled reader will appreciate that a commercially useful protein may, or may not, also have a biological effect on the host cell expressing it (such as a heterologous protein), but that that effect is not the main or sole reason for expressing the protein therein. Commercially useful proteins may include proteins that are useful as metabolites or antibiotics, and the like.
In one embodiment it is preferred that the desired protein (such as a desired heterologous protein) is not β-lactamase. In another embodiment it is preferred that the desired protein (such as a desired heterologous protein) is not antistasin.
However, the reader will appreciate that neither of these provisos exclude genes encoding either β-lactamase or antistasin from being present in a host cell or on a plasmid of the invention, merely that the gene encoding the desired protein (such as a desired heterologous protein) encodes a protein other than β-lactamase and/or antistasin.
Plasmids useful in the practice of the present invention can. unless specified otherwise, be any type of plasmid. For the purposes of the present invention, references to '"plasmids'" may, or may not, also include a reference to other types of vectors. It may be appropriate to choose a suitable plasmid based on the host cell system in which it will be used.
Many plasmids and other vectors are known for the transformation of various expression systems, including systems employing: bacteria (e.g. Bacillus subtilis or
Escherichia coli) transformed with, for example, recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeasts (e.g. Saccharomyces cerevisiae or Pichia pastoris) transformed with, for example, yeast expression vectors; insect cell systems transformed with, for example, viral expression vectors (e.g. baculovirus); plant cell systems transfected with, for example viral or bacterial expression vectors; animal cell systems, either in cell culture, transgenic or as gene therapy, transfected with, for example, adenovirus expression vectors.
Typical procaryotic vector plasmids are: pUC18, pUC19; pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA, USA): p7>c99A, pKK223-3, pKK233-3, pDR540 and pRIT5 available from Pharmacia (Piscataway, NJ, USA); pBS vectors, Phagescript vectors, Bluescript vectors. pNH8A, pNH16A, pNHI SA. pNH46A available from Stratagene Cloning Systems (La JoIIa, CA 92037, USA).
A typical mammalian cell vector plasmid is pSλ^L available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
Useful yeast plasmid vectors include the 2μm-family plasmids (as described above), as well as pRS403-406 and pRS413-416 which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPJ, LEU2 and ORAS. Plasmids pRS413-416 are Yeast Centromere plasmids (YCps). Other Yips and YCps plasmids may also be used.
Plasmids for use in any aspect of the present invention can be prepared by modifying plasmids, such as 2μm-family plasmids, known in the art by inserting the required sequences (for example, one or more genes encoding chaperones and/or one or more genes encoding a heterologous protein) using techniques well known in the art such as are described in by Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2001, 3rd edition, the contents of which are incorporated herein by reference. For example, one such method involves ligation via cohesive ends. Compatible cohesive ends can be generated on a DNA fragment for insertion and plasmid by the action of suitable restriction enzymes. These ends will rapidly anneal through complementary base pairing and remaining nicks can be closed by the action of DNA ligase.
A further method uses synthetic double stranded oligonucleotide linkers and adaptors. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E.coli DNA polymerase I which remove protruding 3' termini and fill in recessed 3 ' ends. Synthetic linkers and pieces of blunt-ended double-stranded DNA, which contain recognition sequences for defined restriction enzymes, can be ligated to blunt-ended DNA fragments by T4 DNA ligase. They are subsequently digested with appropriate restriction enzymes to create cohesive ends and ligated to an expression vector with compatible termini. Adaptors are also chemically synthesised DNA fragments which contain one blunt end used for ligation but which also possess one preformed cohesive end. Alternath'ely a DNA fragment or DNA fragments can be ligated together by the action of DNA ligase in the presence or absence of one or more synthetic double stranded oligonucleotides optionally containing cohesive ends.
Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including Sigma-Genosys Ltd, London Road, Pampisford, Cambridge, United Kingdom.
Appropriate insertion sites in plasmids (such as 2μm-family plasmids) include, but are not limited to, those discussed above.
Host cells
The present invention also provides a host cell comprising recombinant genes and/or plasmid according to any aspect of the present invention. The host cell may be any type of cell. Many suitable host cell expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Pichia pastoris and Kluyveromyces lactis), filamentous fungi (for example Aspergillus), plant cells, whole plants, animal cells and insect cells. Bacterial and yeast host cells may, or may not, be preferred. Bacterial host cells may be useful for cloning purposes. Yeast host cells may be useful for expression of genes present in the plasmid.
In one embodiment the host cell may. or may not. be a yeast cell, such as a member of the Saccharomyces, Kluyveromyces, Arxula, YarroM'ia, Candida, Schi∑osaccharomyces, Debaryomyces, Xanthophyllomyces, Geothchum, Ashbya, Hortaea, Schwanniomyces, Tήchosporon, Xanthophyllomyces, or Pichia genus. Yeast such Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Pichia membranaefaciens, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha (also known as Pichia augusta), Arxula adeninivorans, Yarrowia lipolytica, Candida boidinii Candida utilis, Schizosaccharomyces pombe may, or may not. be preferred. Other suitable yeast may, or may not, include Debaryomyces hansenii, Xanthophyllomyces dendrorhous, Geotrichum candidum, Ashbya gossypii, Hortaea werneckii, SchM'anniomyces occidentalis, Trichosporon domesticum, and/or Xanthophyllomyces dendrorhous,
It is may, or may not, be particularly advantageous to use a yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins, for instance by disruption of the gene coding sequence, as discussed in WO 2004/083245, the contents of which are incorporated herein by reference.
In another embodiment the host cell may, or may not, be an animal cell. For example, the animal cell may, or may not, be a mammalian cell, such as a human cell type. The host cell type may. or may not, be selected for compatibility with a plasmid type being used. Plasmids obtained from one yeast type can be maintained in other yeast types (Me el al, 1991 , Gene, 108(1), 139-144; lrie el al, 1991 , MoI. Gen. Genet , 225(2). 257-265). For example. pSRl from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae. Optionally, the host cell is compatible with a 2μm-family plasmid (see above for a full description of the following plasmids). For example, where the plasmid is based on pSRl, pSB3 or pSB4 then a suitable yeast cell is Zygosaccharomyces rouxii; where the plasmid is based on pSBl or pSB2 then a suitable yeast cell is Zygosaccharomyces bailli; where the plasmid is based on pSMl then a suitable yeast cell is Zygosaccharomyces fermentati; where the plasmid is based on pKDl then a suitable yeast cell is Kluyveromyces drosophilarum; where the plasmid is based on pPMl then a suitable yeast cell is Pichia membranaefaciens; where the plasmid is based on the 2μm plasmid then a suitable yeast cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis. It is particularly preferred that the plasmid is based on the 2μm plasmid and the yeast cell is Saccharomyces cerevisiae.
A 2μm-family plasmid of the invention can be said to be "based on" a naturally occurring plasmid if it comprises one, two or preferably three of the genes FLP, REPl and REP 2 having sequences derived from that naturally occurring plasmid.
It may, or may not, be particularly advantageous to use a yeast deficient in one or more protein mannosyl transferases involved in O-glycosylation of proteins, for instance by disruption of the gene coding sequence.
Recombinantly expressed proteins can be subject to undesirable post-translational modifications by the producing host cell. For example, the albumin protein sequence does not contain any sites for N-linked glycosylation and has not been reported to be modified, in nature, by 0-linked glycosylation. However, it has been found that recombinant human albumin ("rHA") produced in a number of yeast species can be modified by O-linked glycosylation; generally involving mannose. The mannosylated albumin is able to bind to the lectin Concanavalin A. The amount of mannosylated albumin produced by the yeast can be reduced by using a yeast strain deficient in one or more of the PMT genes (WO 94/04687). The most convenient way of achieving this is to create a yeast which has a defect in its genome such that a reduced level of one of the Pmt proteins is produced. For example, there may, or may not. be a deletion, insertion or transposition in the coding sequence or the regulatory regions (or in another gene regulating the expression of one of the PMT genes) such that little or no Pmt protein is produced. Alternatively, the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
If a yeast other than S. cerevisiae is used, disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. in Pichia past oris or Kluyveromyces lactis. The sequence of PMTl (or any other PKdT gene) isolated from S. cerevisiae may, or may not, be used for the identification or disruption of genes encoding similar enzymatic activities in other fungal species.
The cloning of the PMTl homologue of Kluyveromyces lactis is described in WO
94/04687.
The yeast may, or may not, have a deletion of the HSP 150 and/or YAPS genes as taught respectively in WO 95/33833 and WO 95/23857.
A plasmid as defined above, may, or may not, be introduced into a host through standard techniques. With regard to transformation of procaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad ScL USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 rd Ed. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in
Sherman et al (1986) Methods In Yeast Genetics, A Laboratoiy Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
Methods for the transformation of S. cerevisiae are taught generally in EP 251 IAA, EP 258 067 and WO 90/01063, all of which are incorporated herein by reference. With regard to vertebrate cells, reagents useful in transfecting such cells, for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc.. Gaithersburg, MD 20877, USA.
Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cell, bacterial cells and vertebrate cells. ' Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.
Generally, where a plasmid is used, it will transform not all of the hosts and it will therefore be necessary to select for transformed host cells. Thus, a plasmid may, or may not, comprise a selectable marker, including but not limited to bacterial selectable marker and/or a yeast selectable marker. A typical bacterial selectable marker is the β -lactamase gene although many others are known in the art. Yeast selectable marker include LEU2, TRPl, HIS3, HIS4, URA3, URA5, SFAl, ADE2, METIS, LYS5, LYS2, ILV2, FBAl, PSEl, PDIl and PGKl. Those skilled in the art will appreciate that any gene whose chromosomal deletion or inactivation results in an inviable host, so called "essential" genes, can be used as a selective marker if a functional gene is provided on the plasmid. as demonstrated for PGKl in a pgkl yeast strain (Piper and Curran, 1990, Curr. Genet. 17. 1 19). Suitable "essential" genes can be found within the Stanford Genome Database (SGD), http:://db. yeastgenome.org). Any "essential" gene product (e.g. a product of one of the PDIl, PSEl, PGKl or FBAl genes, and others described elsewhere in this application) which, when deleted or inactivated, does not result in an auxotrophic (biosynthetic) requirement, can be used as a selectable marker on a plasmid in a host cell that, in the absence of the plasmid, is unable to produce that gene product, to achieve increased plasmid stability without the disadvantage of requiring the cell to be cultured under specific selective conditions. By "auxotrophic (biosynthetic) requirement" we include a deficiency which can be complemented by nutrient and other additions or modifications to the growth medium. Cells unable to express functional Pgklp or Fbalp can, however, be complemented by certain additions to growth media and such gene products may not be preferred "essential proteins" according to the present invention. Therefore, preferred "essential marker genes" in the context of the present invention are those that, when deleted or inactivated in a host cell, result in a deficiency which cannot be complemented by any additions or modifications to the growth medium, expect where those additions or modifications are, for example, a polynucleotide, that can restore the ability of the host cell to express the product of the "essential" marker gene, or the product of the "essential" marker gene itself.
Accordingly, a plasmid as provided by, for use in a method of, or comprised in a host cell of, the present invention may, or may not, comprise more than one selectable marker.
One selection technique involves incorporating into the expression vector a DNA sequence marker, with any necessary control elements, that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eucaryotic cell culture, and tetracyclin, kanamycin or ampicillin (i.e. β-lactamase) resistance genes for culturing in E.coli and other bacteria. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
Another method of identifying successfully transformed cells involves growing the cells resulting from the introduction of the plasmid, optionally to allow the expression of a recombinant polypeptide (i.e. where a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is not naturally produced by the host). Cells can be harvested and lysed and their DNA or RNA content examined for the presence of the recombinant sequence using a method such as that described by Southern (1975) J. MoJ. Biol. 98, 503 or Berent el al (1985) Biotech. 3, 208 or other methods of DNA and RNA analysis common in the art. Alternatively, the presence of a polypeptide in the supernatant of a culture of a transformed cell can be detected using antibodies.
In addition to directly assaj'ing for the presence of recombinant DNA. successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein. For example, cells successfully transformed with an expression vector produce proteins displaying appropriate antigenicity. Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
Thus, in addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, optionally a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. Alternatively, transformed cells may, or may not, represent an industrially/commercially or pharmaceutically useful product and can be used without further purification or can be purified from a culture medium and optionally formulated with a carrier or diluent in a manner appropriate to their intended industrial/commercial or pharmaceutical use, and optionally packaged and presented in a manner suitable for that use. For example, whole cells could be immobilised; or used to spray a cell culture directly on to/into a process, crop or other desired target.
Similarly, whole cell, such as yeast cells can be used as capsules for a huge variety of applications, such as fragrances, flavours and pharmaceuticals.
Transfoπned host cells may, or may not, be cultured for a sufficient time and under appropriate conditions known to those skilled in the art, and in view of the teachings disclosed herein, to permit the expression of one or more recombinant chaperones and a desired protein (such as a desired heterologous protein). The culture medium may, or may not. be non-selective or may. or may not place a selective pressure on the maintenance of the plasmid.
The thus produced desired protein (such as a desired heterologous protein) may. or may not, be present intracellular!}' or. if secreted, in the culture medium and/or periplasmic space of the host cell.
Protein Recovery and Formulation
The step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium optionally comprises cell immobilization, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilization, separation and/or breakage.
Cell immobilization techniques, such as encasing the cells using calcium alginate bead, are well known in the art. Similarly, cell separation techniques, such as centrifugation, filtration (e.g. cross-flow filtration, expanded bed chromatography and the like are well known in the art. Likewise, methods of cell breakage, including beadmilling, sonication, enzymatic exposure and the like are well known in the art.
The at least one other purification step may be any other step suitable for protein purification known in the art. For example purification techniques for the recovery of recombinantly expressed albumin have been disclosed in: WO 92/04367, removal of matrix-derived dye; EP 464 590, removal of yeast-derived colorants; EP 319 067, alkaline precipitation and subsequent application of the albumin to a lipophilic phase; and WO 96/37515, US 5 728 553 and WO 00/44772, which describe complete purification processes; all of which are incorporated herein by reference. Proteins other than albumin may be purified from the culture medium by any technique that has been found to be useful for purifying such proteins.
Suitable methods include ammonium sulphate or ethanol precipitation, acid or solvent extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography ("HPLC"), reverse phase HPLC, conductivity adjustment and the like.
In one embodiment, any one or more of the above mentioned techniques may, or may not, be used to further purify the thus isolated protein to a commercially or industrially acceptable level of purity. By commercially or industrially acceptable level of purity, we include the provision of the protein at a concentration of at least 0.01 g.L"1, 0.02 g.L"1, 0.03 g.L'1, 0.04 g.L"1, 0.05 g.L"',0.06 g.L"',0.07 g.1/1, 0.08 g.L" \ 0.09 g.L"1, 0.1 g.L"1, 0.2 g.L"1, 0.3 g.L"1, 0.4 g.L"1, 0.5 g.L"1, 0.6 g.L"1, 0.7 g.L"1, 0.8 g.L"1, 0.9 g.1/1, 1 g.L"1, 2 g.L"1, 3 g.L"1, 4
Figure imgf000089_0001
5 g.L"1, 6 g.L"1, 7 g.L"1, 8 g.L"1, 9 g.L" ', 10 g.L"1, 15 g.L"1, 20 g.1/1, 25 g.L"1, 30 g.L"1, 40 g.lΛsO g.I/1, 60 g.L'1, 70 g.L"1, 70 g.L"1, 90 g.L"1, 100 g.L"1, 150 g.L"1, 200 g.I/1 ,250 g.L"1, 300 g.L"1, 350 g.L"1, 400 g.L"1, 500 g.L"1, 600 g.L"1, 700 g.L"1, 800 g.L"1, 900 g.L"1, 1000 g.L"1, or more.
It is preferred that the desired protein (such as a desired heterologous protein) is purified to achieve a pharmaceutically acceptable level of purity. A protein has a pharmaceutically acceptable level of purity if it is essentially pyrogen free and can be administered in a pharmaceutically efficacious amount without causing medical effects not associated with the activity of the protein.
The resulting desired protein (such as a desired heterologous protein) may, or may not, be used for any of its known utilities, which, in the case of albumin, include i.v. administration to patients to treat severe bums, shock and blood loss, supplementing culture media, and as an excipient in formulations of other proteins.
Although it is possible for a therapeutically, diagnostically, industrially. domestically or nutritionally useful desired protein (such as a desired heterologous protein) obtained by a process of the of the invention to be presented or administered alone, it is preferable to present it as a formulation (such as a pharmaceutical formulation, particularly in the case of therapeutically and/or diagnostically useful proteins), together with one or more acceptable carriers or diluents. The carrier(s) or diluent(s) must be '"acceptable" in the sense of being compatible with the desired protein and, where the formulation is intended for administration to a recipient, then not deleterious to the recipient thereof. Typically, the carriers or diluents will be water or saline which will be sterile and pyrogen free.
Optionally the thus formulated protein will be presented in a unit dosage form, such as in the form of a tablet, capsule, injectable solution or the like.
In a sixth aspect of the present invention there is provided a method for producing a desired protein (such as a desired heterologous protein), such as a desired protein as defined above for an earlier aspect of the present invention, comprising: providing a host cell comprising a first recombinant gene encoding the protein comprising the sequence of 0rm2p or a variant thereof and a second gene, optionally a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), optionally with the proviso that the first and second genes are not both present within the host cell on the same 2μm-family plasmid; and culturing the host cell in a culture medium under conditions that allow the expression of the first and second genes. The method may, or may not, further comprise the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium; and optionally, lyophilising the thus purified protein; and optionally formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent; and optionally presenting the thus formulated protein in a unit dosage form.
In the manner discussed above, the host cell may, or may not, further comprise a further recombinant gene encoding a protein comprising the sequence of an alternative chaperone toθrm2p or a variant thereof.
Either or both of the first and second genes in the sixth aspect of the invention may or may not be recombinant genes that are expressed from a plasmid, and optionally from the same plasmid, provided that where both genes are expressed from the same plasmid then that plasmid is not a 2μm-family plasmid. A further recombinant gene encoding a protein comprising the sequence of an alternative chaperone to 0rm2p or a variant thereof may, or may not, also be expressed from a plasmid, optionally from the same plasmid as either or both of the first and second recombinant genes. Except where both of the first and second genes are recombinant genes that are co-expressed from the same plasmid then either one may, or may not, be individually expressed from a 2μm-family plasmid, such as the 2μm plasmid. Alternatively, one or both of the first and second genes of the sixth aspect of the invention may, or may not, be integrated into the chromosome of the host cell. The further recombinant gene encoding a protein comprising the sequence of an alternative chaperone to 0rm2p or a variant thereof may, or may not, be integrated into the chromosome of the host cell, irrespective of whether or not the first and second genes are expressed from a plasmid or are chromosomally integrated.
The present invention also provides, in a seventh aspect, a host cell as defined above in respect of the sixth aspect, which host cell comprises a first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof and a second gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein), optionally with the proviso that the first and second genes are not present within the host cell on the same 2μm-family plasmid.
The present invention also provides, in an eighth aspect, for the use of a nucleic acid sequence encoding the protein 0rm2p or a variant thereof to increase the production, in a host cell (such as a host cell as defined above), of a desired protein (such as a desired heterologous protein) encoded by a gene, such as a recombinant gene, in the host cell by co-expression of the nucleic acid sequence and the gene within the host cell (but optionally not including co-expression of these genes from the same 2μm-family plasmid). Either or both of the nucleic acid sequence and the gene encoding the desired protein may, or may not, be expressed from a plasmid within the host cell, and optionally from the same plasmid. In the manner discussed above, the host cell may, or may not, further comprise a recombinant gene encoding an alternative chaperone to 0rm2p or a variant thereof, which may, or may not, be located on a plasmid within the host cell, optionally on the same plasmid as either or both of the nucleic acid sequence and a gene encoding the desired protein. Suitable plasmids include a 2μm-family plasmid, such as the 2μm plasmid, as discussed above.
In a ninth aspect of the present invention there is also provided the use of a plasmid as an expression vector to increase the production of a heterologous protein by providing a recombinant gene encoding the heterologous protein and a gene encoding Orni2p or a variant thereof on the same plasmid, optionally with the proviso that the plasmid is not a 2μm-family plasmid. The plasmid may, or may not, further comprise a gene encoding an alternative chaperone to 0rm2p or a variant thereof in the manner discussed above.
Accordingly, in a tenth aspect, the present invention also provides a plasmid, optionally an expression plasmid, comprising a first gene encoding the protein Orm2p or a variant or fragment thereof and a second gene encoding a heterologous protein, as discussed above, optionally with the proλ'iso that the plasmid is not a 2μm-family plasmid. The plasmid may. or may not, further comprise a third gene encoding an alternative chaperone to 0rm2p or a variant thereof. In a preferred embodiment, the third gene encodes a protein comprising the sequence of protein disulphide isomerase.
We have also demonstrated that a plasmid-borne gene encoding a protein comprising the sequence of an "essential" protein can be used to stably maintain the plasmid in a host cell that, in the absence of the plasmid. does not produce the "essential" protein. This has the advantage of ensuring the genetic stability of the organism in the chosen culture conditions, and thereby improving the reproducibility and reliability of individual cultures.- and furthermore enables prolonged culture without reduced productivity due to plasmid loss.
A preferred "essential" protein is a chaperone which may or may not provide the further advantage that, as well as acting as a selectable marker to increase plasmid stability, its expression simultaneously increases the expression of one or more desired proteins, such as a heterologous protein encoded by a recombinant gene, within the host cell. This system is advantageous because it allows the user to minimise the number of recombinant genes that need to be carried by a plasmid. For example, typical prior art plasmids carry marker genes (such as those as described above) that enable the plasmid to be stably maintained during host cell culturing process. Such marker genes need to be retained on the plasmid in addition to any further genes that are required to achieve a desired effect. However, the ability of plasmids to incorporate exogenous DNA sequences is -limited and it is therefore advantageous to minimise the number of sequence insertions required to achieve a desired effect. Moreover, some marker genes (such as auxotrophic marker genes) require the culturing process to be conducted under specific conditions in order to obtain the effect of the marker gene. Such specific conditions may not be optimal for cell growth or protein production, or may require inefficient or unduly expensive growth systems to be used.
Thus, it is possible to use a recombinant gene that encodes a protein comprising the sequence of an "essential" protein as a plasmid-borne gene to increase plasmid stability, where the plasmid is present within a cell that, in the absence of the plasmid. is unable to produce the "essential" protein. It will be appreciated that the question of whether or not a protein is "essential" will depend on the system in which it is use; it is possible that a protein that is not "essential" in one host organism might become "essential" when one or more other genes is deleted, disrupted, inactivated, modified or affected in that same host, and thereby be used as an "essential" plasmid-borne gene, as described above; likewise whether or not a protein is "essential" may depend on certain physical conditions, such as pH, temperature and/or oxygen levels under which the host cell is cultured..
It is preferred that the "essential protein" is one that, when its encoding gene(s) in a host cell are deleted or inactivated, does not result in the host cell developing an auxotrophic (biosynthetic) requirement. By "auxotrophic (biosynthetic) requirement" we include a deficiency that can be complemented by additions or modifications to the growth medium, in particular additions of, or modifications to, the nutrient composition of the growth medium. Thus, the "essential protein" would be an auxotrophic marker protein if the inactivation of its encoding gene, in a host cell, resulted in the production of an auxotrophic mutant, i.e. a mutant organism that, in order to grow and survive, requires a particular additional nutrient that the normal (unmutated strain) does not - it is preferred that the "essential protein" is not an auxotrophic marker protein. Therefore, an "essential marker gene" which encodes an "essential protein", in the context of the present invention is one that, when deleted or inactivated in a host cell, results in a deficiency which cannot be complemented by additions or modifications, typically nutrient additions or modifications, to the growth medium, expect where those additions or modifications are, for example, a polynucleotide, that can restore the ability of the host cell to express the product of the "essential" marker gene, or the product of the "essential" marker gene itself. In other words, it may, or may not. be preferred if the ''essential protein" is not a protein that, in nature, is involved in the metabolic conversion of nutrients by a host cell. The advantage of this system is that the '"essential marker gene" can be used as a selectable marker on a plasmid in host cell that, in the absence of the plasmid. is unable to produce that gene product, to achieve increased plasmid stability without the disadvantage of requiring the cell to be cultured under specific selective (e.g. selective nutrient) conditions. Therefore, the host cell can be cultured under conditions that do not have to be adapted for any particular marker gene, without losing plasmid stability. For example, host cells produced using this system can be cultured in non-selective media such as complex or rich media, and under non-selective growth conditions (e.g. such as pH, temperature and/or oxygen levels), which may be more economical, and/or more supportive growth media/conditions, than the minimal media and/or specifically adapted growth conditions that are commonly used to give auxotrophic, and other, marker genes their effect.
The cell may, or may not, for example, have the endogenous copy (or copies) of the gene (or genes) encoding the "essential" protein deleted or otherwise inactivated.
It is particularly preferred if the "essential protein" is an "essential" chaperone, as this can provide the dual advantage of improving plasmid stability without the need for selective growth conditions and increasing the production of desired proteins, such as endogenously encoded or a heterologous proteins, in the host cell. This system also has the advantage that it minimises the number of recombinant genes that need to be carried by the plasmid if one chooses to use over-expression of an "essential" chaperone to increase protein production by the host cell.
Preferred "essential proteins" for use in this aspect of the invention include the '"essential" chaperones encoded by the genes PDIl and PSEl which, when the endogenous gene(s) encoding these proteins are deleted or inactivated in a host cell, do not result in the host cell developing an auxotrophic (biosynthetic) requirement.
Preferred "essential" chaperones are eucaryotic chaperones. especially preferred "essential" chaperones are yeast chaperones. including chaperones comprising the sequence of proteins encoded by a gene selected from CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNSl, EROl (in the absence of diamide), HSPlO., HSP 60, PDIl, CDCSl, KAR2, MGEl, MRSIl, NOBl, SSCl, PSEl, TIM9, PAMl 8 and TCPl.
It is noted that a host cell that is mutated to inactive EROl can be complemented by growth in the presence of the oxidant diamide (Frand & Kaiser, 1998, Molecular Cell, I5 161-170), but diamide is not a "nutrient" addition of the type discussed above in respect of auxotrophic mutations. Diamide is not a commonly used component of growth media and an EROl mutant that is transformed with a plasmid comprising the EROl gene can be grown in rich media without loss of plasmid stability.
Accordingly, in an eleventh aspect, the present invention also provides a host cell comprising a plasmid (such as a plasmid as defined above by any of the previous aspects of the invention), the plasmid comprising a gene that encodes an
"essential" protein, such as a chaperone, wherein, in the absence of the plasmid, the host cell is unable to produce the "essential" protein. Preferably, in the absence of the plasmid, the host cell is inviable. Typically the host cell has been genetically modified to render it unable to produce a functional copy of the
"essential" protein from a chromosomally-encoded (or otherwise endogenous) gene. The host cell may, or may not, further comprise a recombinant gene encoding a heterologous protein, such as those described above in respect of earlier aspects of the invention. The present invention also provides, in a twelfth aspect, a plasmid comprising, as the sole yeast selectable marker, optionally as the sole selectable marker, a gene encoding an "essential" protein, such as an ''essential" chaperone. The plasmid may. or may not, further comprise a gene encoding a heterologous protein. The plasmid may, or may not be a 2μm-family plasmid.
The present invention also provides, in a thirteenth aspect, a method for producing a desired protein (such as a desired heterologous protein) comprising the steps of: providing a host cell comprising a plasmid, the plasmid comprising a gene that encodes an "essential" protein, such as a chaperone, wherein, in the absence of the plasmid, the host cell is unable to produce the "essential" protein and wherein the host cell further comprises a gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein); culturing the host cell in a culture medium under conditions that allow the expression of the "essential" protein and the desired protein; and optionally purifying the thus expressed desired protein from the cultured host cell or the culture medium: and further optionally, lyophilising the thus purified protein. Thus, a host cell used in this method may, or may not, be a host cell according to the eleventh aspect of the invention and/or the host call may, or may not, be transformed with a plasmid according to the twelfth aspect of the invention.
The method may, or may not, further comprise the step of formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form, in the manner discussed above.
In one preferred embodiment, the step of "culturing the host cell in a culture medium under conditions that allow the expression of the "essential" protein and the desired protein" involves culturing the host cell in medium that is not specifically adapted to be selective for the presence of any genes on the plasmid, other than for the presence of the gene encoding the "essential" protein. Thus, in one embodiment, the step of culturing the host cells may. or may not. be performed in non-selective media, such as complex or rich media and/or under conditions (such as pH, temperature and/or oxygen levels) that are not specifically adapted to select for the presence of the '"essential" protein. A medium can be described as non-selective for the purposes of the present situation if it is not specifically adapted to deprive the host cell of a product, typically a nutrient product, that is ordinarily provided to maximise, or otherwise allow, the growth of host cells that have not been modified to prevent the expression of the "essential" protein. For example, a medium (the "test medium") may be a non-selective medium, for the purposes of the present invention if, when one compares plasmid stability in a first host cell type grown in the 'lest medium" to plasmid stability in a second cell type grown in the "test medium" when each cell type is grown for 5, 10, 15, 20, 25 or 30 generations in the "test medium", wherein - (i) the first host cell type is a host cell according to the eleventh aspect of the present invention;
(ii) the second host cell type is a host cell according to the eleventh aspect of the present invention except that it has been modified to restore the ability of the host cell to produce the "essential" protein in the absence of the plasmid (which is not to say that the second host cell type does not contain a plasmid encoding the "essential" protein, just that it can produce the "essential" protein even when the plasmid is not present); then the plasmid stability observed in the second cell type is less than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less of the plasmid stability observed in the first cell type. Thus, in this embodiment, and despite being grown under non-selective conditions, it is preferable for method of the thirteenth aspect of the present invention to produce host cells which display substantially 100% plasmid stability after 5, 10, 15, 20, 25 or 30 generations. A fourteenth aspect of the present invention also provides for the use of a polynucleotide that encodes an "essential" protein (as defined above) to increase the stability of a plasmid in a host cell, particularly under non-selective conditions, by integration of the polynucleotide into the plasmid to produce a modified plasmid. wherein the host cell is unable to produce the '"essential" protein in the absence of the modified plasmid. The increase in stability may. or may not. be at least 1% (i.e. 1.01 times), 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%. 30%, 40%, 50%, 60%, 70%, 80%; 90%, 100% (i.e. 2-fold), 3-fold, 4-fold, 5-fold, 6-fold s, 7-fold, 8-fold, 9-fold, 10-fold or more greater than the level of stability of the unmodified plasmid in the same host cell that has been modified to produce the ''essential'" protein, when grown for at least five generations under non-selective conditions.
In one embodiment of the fourteenth aspect of the present invention, the plasmid may, or may not, additionally comprise a gene that encodes a further desired heterologous protein, such as defined above in respect of earlier aspects of the present invention. In that case, the use may, or may not, be to improve the productivity of the desired heterologous protein, such as when the host cell comprising the plasmid is grown under non-selective conditions.
In one preferred embodiment, the "essential" protein is a chaperone and may, or may not, be used to simultaneously increase the stability of a plasmid in the host cell and increase the ability of the host cell to produce a desired protein product. The desired protein product may, or may not, be an endogenous!}' encoded protein or may, or may not, be a heterologous protein as defined by the earlier aspects of the invention. Where the protein product is a heterologous protein, it may, or may not, be encoded by a recombinant gene that has been integrated into the chromosome of the host cell, or by a gene that is present on a plasmid in the host cell, such as the modified plasmid comprising the polynucleotide that encodes the "essential" protein as defined above. We have also found that the effects of recombinantly-provided chaperones according to the other embodiments of the present invention can be modulated by modifying the promoters that control the expression levels of the chaperone(s). Surprisingly we have found that, in some cases, shorter promoters result in increased expression of a desired protein. Without being bound by theory we believe that this is because the expression of a recombinant chaperone in host cells that already express desired proteins at high levels can cause the cells to overload themselves with desired protein (such as a desired heterologous protein), thereby achieving little or no overall increase in production of the desired protein. In those cases, it may, or may not, be beneficial to provide recombinant chaperone genes with truncated promoters.
Accordingly, in a fifteenth aspect of the present invention there is provided a polynucleotide (such as a plasmid as defined above) comprising the sequence of a promoter operably connected to a coding sequence encoding a chaperone (such as those described above), for use in increasing the expression of a desired protein (such as a desired heterologous protein), such as those described above, in a host cell (such as those described above) by expression of the polynucleotide sequence within the host cell, wherein the promoter is characterised in that it achieves a modified, such as a higher or lower, level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter.
The present invention also provides, in a sixteenth aspect, a method for producing a desired protein (such as a desired heterologous protein) comprising the steps of: providing a host cell comprising a recombinant gene that comprising the sequence of promoter operably connected to a coding sequence encoding a chaperone, the promoter being characterised in that it achieves a lower level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter, and the host cell further comprising a gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein); culturing the host cell under conditions that allow the expression of the chaperone and the desired protein; and optionally purifying the thus expressed desired protein from the cultured host cell or the culture medium; and further optionally, lyophilising the thus purified protein; and optionally further formulating the purified desired protein with a carrier or diluent; and optionally presenting the thus formulated protein in a unit dosage form, in the manner discussed above.
As is apparent from the examples of the present application, the combination of recombinantly expressed PDI and transferrin-based proteins provides a surprisingly high level of transferrin expression. For example, transferrin expression in a system that includes a chromosomally encoded recombinant PDI gene provided a 2-fold increase (compared to a control in which there is no chromosomally encoded recombinant PDI gene). This increase was 5-times greater than an equivalent system comprising a recombinant gene encoding human albumin in place of the recombinant transferrin gene.
The host may be any cell type, such as a procaryotic cell (e.g. bacterial cells such as E. colϊ) or a eucaryotic cell. Preferred eucaryotic cells include fungal cells, such as yeast cells, and mammalian cells. Exemplar}7 yeast cells are discussed above. Exemplary mammalian cells include human cells.
Host cells as described above can be cultured to produce recombinant transferrin- based proteins. The thus produced transferrin-based proteins can be isolated from the culture and purified, optionally to a pharmaceutically acceptable level of purity, for example using techniques known in the art and/or as set out above. Purified transferrin-based proteins may, or may not, be formulated with a pharmaceutically acceptable carrier or diluent and may, or may not, be presented in unit dosage form. The present invention will now be exemplified with reference to the following non-limiting examples and figures.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a map of a typical 2μm plasmid.
Figure 2 shows the results of rocket immunoelectrophoresis (RIE) determination of increased recombinant transferrin (N413Q, N61 IQ) secretion with PDIl over- expression. Cryopreserved yeast stocks were grown for 4-days in 1OmL BMMD shake flask cultures and supernatants were loaded at 5μL per well. Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) was used at 40μL per rocket immunoelectrophoresis gel (5OmL). A = Control strain [pSAC35], duplicate flasks; B = Control strain [pDB2536], duplicate flasks; C = Control strain [pDB2711], neat to 40-fold aqueous dilutions; D = Control strain [pDB2931], duplicate flasks; E = Control strain [pDB2929], neat to 40-fold aqueous dilutions.
Figure 3 shows the results of RIE analysis of recombinant transferrin (N413Q, N611Q) secretion with and without PDIl over-expression. Cryopreserved yeast stocks were grown for 4-days in 1OmL BMMD shake flask cultures and supernatants were loaded at 5μL per well. Duplicate loadings were made of supernatants from two individual cultures of each strain. Goat polyclonal anti- transferrin (human) antiserum (Calbiochem) was used at 40μL per rocket immunoelectrophoresis gel (5OmL). A = Control strain [pSAC35]; B = Control strain [pDB2536]; C = Control strain [pDB2711]; D = Control strain [pDB2931]; E = Control strain [pDB2929].
Figure 4 shows the results of SDS-PAGE analysis of recombinant transferrin secretion with and without PDIl over-expression. BMMD shake flask cultures were grown for 4-days and 10μL supernatant analysed on non-reducing SDS- PAGE (4-12% NuP AGE®, MOPS buffer, inVitrogen) with GelCode® Blue reagent (Pierce). 1 = SeeBlue Plus2 Markers (InVitrogen). 2 = pDB2536; 3 = pDB2536: 4 = pDB2711 ; 5 = pDB2711; 6 = pDB2931; 7 = pDB2931 ; 8 = pDB2929; 9 = pDB2929; 10 = pSAC35 control.
Figure 5 shows RJE analysis of recombinant transferrin secretion from S. cerevisiae strains with an additional integrated copy of PDIl . 5-day BMMD shake flask culture supernatants were loaded at 5mL per well. Strains contained: 1) pSAC35 (negative control); 2) pDB2536 (recombinant non-glycosylated transferrin (N413Q, N611Q)) or 3) pDB2506 (same as plasmid pDB2536 but the transferrin ORF encodes transferrin without the N→Q mutations at positions 413 and 611, i.e. recombinant glycosylated transferrin). Each well contained a sample derived from an individual transformant. Standards were human plasma holo- transferrin (Calbiochem) at 100, 50, 20, 10, 5 and 2mg.lΛ
Figure 6 shows RIE analysis of recombinant transferrin secretion from Strain A [pDB2536] and Strain A [pDB2506] grown in shake flask culture. 5-day BMMD or YEPD shake flask culture supernatants were loaded in duplicate at 5mL per well.
Figure 7 shows SDS-PAGE analysis of recombinant transferrin secreted from Strain A [pDB2536] and Strain A [pDB2506] grown in shake flask culture. Cultures were grown for 5 -days in BMMD and 3OmL supernatants analysed on SDS-PAGE (4-12% NuP AGE™, MOPS Buffer, InVitrogen) stained with GelCode, Blue Reagent (Pierce). 1) Strain A [pDB2536] transformant 1 ; 2) Strain A [pDB2536] transfonnant 2; 3) Strain A [pSAC35] control; 4) Strain A [pDB2506] transformant 1; 5) SeeBlue, Plus2 Protein Standards (approximate molecular weights only). Figure 8 shows RIE of recombinant transferrin secreted from 5 cerevisiae strains with different PDIl copy numbers. 3 -day BMMD shake flask culture supernatants were loaded at 5mL per well. Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) was used at 3OmL per rocket Immunoelectrophoresis gel (5OmL). (A) supernatant from S. cerevisiae control strain [pDB2711] or [pDB2712]; (B) supernatant from Strain A [pDB2536]; (C) supernatant from control strain [pDB2536].
Figure 9 shows SDS-PAGE analysis of recombinant transferrin secreted from S. cerevisiae strains with different PDIl copy numbers. 4-12% NuPAGE reducing gel run with MOPS buffer (InVitrogen) after loading with 3OmL of 3-day BMMD shake flask culture supernatant per lane; (lane 1) supernatant from control strain [pDB2536]; (lane 2) supernatant from Strain A [pDB2536]; (lanes 3-6) supernatant from control strain [pDB271 1] or [pDB2712]; (lane 7) molecular weight markers (SeeBlue Plus2. InVitrogen).
Figure 10 shows RIE of recombinant transferrin secreted from different S. cerevisiae strains with and without additional PDIl gene co-expression. 1OmL YEPD shake flasks were inoculated with yeast and incubated for 4-days at 30°C. 5μL culture supernatant loaded per well of a rocket Immunoelectrophoresis gel. Plasma Tf standards concentrations are in μg/mL. 20μL goat anti-Tf / 5OmL agragose. Precipin was stained with Coomassie blue.
Figure 11 shows RIE analysis of rHA expression in different S. cerevisiae strains when co-expressed with PDIl genes having different length promoters. 1OmL
YEPD shake flasks were inoculated with yeast and incubated for 4-days at 30°C.
4μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in μg/mL. 400μL goat anti-HA (Sigma product
A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue. Figure 12 shows RIE analysis of rHA expression in different S. cerevisiae strains when co-expressed with PDIl genes having different length promoters. 1OmL YEPD shake flasks were inoculated with yeast and incubated for 4-days at 300C. 4μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in μg/mL. 400μL goat anti-HA (Sigma product A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue.
Figure 13 shows RIE analysis of rHA fusion proteins with and without co- expressed recombinant PDIl. 1OmL BMMD shake flasks were inoculated with YBX7 transformed with albumin fusion expression plasmids and incubated for 4- days at 300C. 4μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in μg/mL. 200μL goat anti-HA (Sigma product A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue.
Figure 14 shows SDS-PAGE analysis of recombinant albumin fusion secretion with and without PDIl present on the expression plasmid. 1OmL BMMD shake flasks were inoculated with yeast and incubated for 4-days at 300C. 200rpm. 30μL supernatant analysed on non-reducing SDS-PAGE (4-12% NuPAGE®, MES buffer, InVitrogen) with GelCode® Blue reagent (Pierce). 1 = SeeBlue Plus2 Markers (InVitrogen); 2 = 1 μg rHA; 3 = angiostatin-rHA; 4 = angiostatin-rHA + PDU\ 5 = endostatin-rHA; 6 = endostatin-rHA + PDIl; 7 = DX-890-(GGS)4GG- rHA; 8 = DX-890-(GGS)4GG-rHA + PDIl; 9 = DPI-14-(GGS)4GG-rHA; 10 = DPI-14-(GGS)4GG-rHA + PDIl; 11 = Axokine™ (CNTFAX15)-(GGS)4GG-ΓHA (Lambert et al, 2001, Proc. Natl. Acad. Sci. USA, 98, 4652-4657); 12 = Axokine™ (CNTF Axi s) -(GGS)4GG-IHA + PDIl . Figure 15 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with 0RM2 co-expression from a 2μm-based plasmid. Four day shake flask culture supernantants were loaded at 5μl per well. Standards were human plasma holo-transferrin (Calbiochem), at 25, 20, 15. 10, 5 μg/ml, loaded 5μl per well. Goat polyclonal anti -transferrin (human) antiserum (Calbiochem) used at 20 μl per rocket immunoelectrophoresis gel (50 ml).
Figure 16 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with PSEl co-expression from a 2μm-based plasmid. Four day shake flask culture supernantants were loaded at 5μl per well. Standards were human plasma holo-transferrin (Calbiochem), at 25. 20, 15, 10. 5 μg/ml, loaded 5μl per well. Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) used at 20μl per rocket immunoelectrophoresis gel (50 ml).
Figure 17 shows RIE analysis demonstrating increased transferrin secretion from S. cerevisiae with SSAl co-expression from a 2μm-based plasmid. Four day shake flask culture supernantants were loaded at 5μl per well. Standards were human plasma holo-transferrin (Calbiochem). at 25, 20, 15, 10, 5 μg/ml, loaded 5μl per well. Goat polyclonal anti-transferrin (human) antiserum (Calbiochem) used at 20 μl per rocket immunoelectrophoresis gel (50 ml).
Figure 18 shows the results of RIE. 1OmL YEPD shake flasks were inoculated with DXYl trplΔ [pDB2976], DXYl trplΔ [pDB2977], DXYl trplΔ [pDB2978], DXYl trplΔ [pDB2979], DXYl trplΔ [pDB2980] or DXYl trplΔ [pDB2981] transformed to tryptophan prototrophy with a 1.41kb NotVPstl pdiL. TRPl disrupting DNA fragment was isolated from pDB3078. Transformants were grown for 4-days at 300C, 200rpm. 4μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in μg/mL. 700μL goat anti-HA (Sigma product A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue. Isolates selected for further analysis are indicated (*).
Figure 19 shows the results of RIE. 1OmL YEPD shake flasks were inoculated with DXYl [pDB2244], DXYl [pDB2976], DXYl trplΔ pdil::TRPl [pDB2976], DXYl [pDB2978], DXYl trplΛ pdilr. TRPl [pDB2978], DXYl [pDB2980], DXYl trplA pdilr. TRPl [pDB2980], DXYl [pDB2977], DXYl trplA pdilr. TRPl [pDB2977], DXYl [pDB2979] DXYl trplΔ pdilrTRPl [pDB2979], DXYl [pDB2981] and DXYl trplΔ pdilr TRPl [pDB2981], and were grown for 4-days at 300C, 200rpm. 4μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. rHA standards concentrations are in μg/mL. 800μL goat anti-HA (Sigma product A-1151 resuspended in 5mL water) /5OmL agarose. Precipin was stained with Coomassie blue. Isolates selected for further analysis are indicated (*)
Figure 20 shows a sequence alignment of the SKQ2n and S288c gene sequences with long promoters, as described in Example 6.
Figures 21 to 33 show various plasmid maps.
Figure 34 shows Rocket Immunoelectrophoresis of YEPD shake flask culture supernatants from DXYl and DXYl Δtrpl pdilrTRPl containing pDB3175 to pDB3178. 1OmL YEPD shake flasks were inoculated with DXYl [pAYE316], DXYl [pDB3175], DXYl [pDB3176], DXYl [pDB3177], DXYl [pDB3178], DXYl Δtrpl pdilrTRPl [pDB3175], DXYl Δtrpl pdilr TRPl [pDB3176], DXYl Δtrpl pdilr. TRPl [pDB3177], and DXYl Δtrpl pdilrTRPl [pDB3178] were grown for 4-days at 3O°C. 200rpm. 5μL culture supernatant was loaded per well of a 5OmL rocket immunoelectrophoresis gel in triplicate. rHA standard concentrations were 300, 200: 150, 100 and 50 μg/mL. 600μL goat anti-HSA (Sigma product A-1 151 resuspended in 5mL water) per 5OmL agarose gel. Precipitin was stained with Coomassie blue.
Figure 35 shows Rocket Immunoelectrophoresis of YEPD shake flask culture supernatants from DXYl and DXYl Atrpl pdil::TRPl containing pDB3179 to pDB3182. 1OmL YEPD shake flasks were inoculated with DXYl [pDB2931], DXYl [pDB3179], DXYl [pDB3180], DXYl [pDB3181], DXYl [pDB3182], DXYl Δtrpl pdil::TRPl [pDB3179], DXYl Atrpl pdilr. TRPl [pDB3180], DXYl Atrpl pdilr. TRPl [pDB3181], and DXYl Atrpl pdil::TRPl [pDB3182] were grown for 4-days at 30°C, 200rpm. 5μL culture supernatant was loaded per well of a 5OmL rocket immunoelectrophoresis gel in triplicate. Plasma transferrin standard concentrations were 100, 50, 20, 10 and 5 μg/mL. 40μL goat polyclonal anti human transferrin antiserum (Calbiochem) was used per 5OmL agarose gel. Precipitin was stained with Coomassie blue.
EXAMPLES
Two types of expression cassette haλ'e been used to exemplify secretion of a recombinant human transferrin mutant (N413Q, N611Q) from S. cerevisiae. One type uses a modified HSA(pre)/MFαl(pro) leader sequence (named the "modified fusion leader" sequence). The second type of expression cassette uses only the modified HSA(pre) leader sequence.
The 24 amino acid sequence of the "modified fusion leader" is MKWVFIVSILFLFSSAYSRSLDKR.
The 18 amino acid sequence of the modified HSA(pre) leader sequence is MKWVFIVSILFLFSSAYS. Transferrin (N413Q, N611Q) expression using these two cassettes has been studied in S. cerevisiae using the 2μm expression vector with and without an additional copy of the S. cerevisiae PDl gene. PDIl .
EXAMPLE 1
Construction of expression plasmids
Plasmids pDB2515, pDB2529, pDB2536, pDB2688, pDB2690, pDB2711, pDB2921, pDB2928, pDB2929, pDB2930, pDB293L pDB2932 and pDB2690 were constructed as described in Example 1 of WO 2005/061718. the contents of which are incorporated herein by reference.
EXAMPLE 2
Expression of transferrin
A S. cerevisiae control strain was transformed to leucine prototrophy with all the transferrin (N413Q, N611Q) expression plasmids, and cryopreserved stocks were prepared.
Strains were grown for four days at 30°C in 1OmL BMMD cultures in 5OmL conical flasks shaken at 200rpm. The titres of recombinant transferrin secreted into the culture supernatants were compared by rocket Immunoelectrophoresis (RIE as described in Weeke, B., 1976, "Rocket Immunoelectrophoresis" In N. H. Azelsen, J. Kroll, and B. Weeke [eds.], A manual of quantitative immunoelectrophoresis. Methods and applications. Universitetsforlaget, Oslo, Norway), reverse phase high performance liquid chromatography (RP-HPLC) (Table 1), and non-reducing SDS polyacryl amide electrophoresis stained with colloidal Coomassie blue stain (SDS-PAGE). The increase in recombinant transferrin secreted when S. cerevisiae PDIl was over-expressed was estimated to be greater than 10-fold.
By RP-HPLC analysis (using the method described in Example 2 of WO 2005/061718, the contents of which are incorporated herein by reference) the increase in transferrin secretion was determined to be 18-fold for the modified fusion leader sequence and 15-fold for the modified HSA-pre leader sequence (Table 1).
Figure 4 shows an SDS-PAGE comparison of the recombinant transferrin secreted by S. cerevisiae strains with and without additional PDIl expression.
Table 1:
Figure imgf000111_0001
RIE analysis indicated that the increased transferrin secretion in the presence of additional copies oiPDIl was approximately 15-fold (Figure 2). By RIE analysis the increase appeared slightly larger for the modified HSA-pre leader sequence than for the modified fusion leader sequence (Figure 3). EXAMPLE 3
Chromosomal over-expression of PDI
S. cerevisiae Strain A was selected to investigate the secretion of recombinant glycosylated transferrin expression from plasmid pDB2506 and recombinant non- glycosylated transferrin (N413Q, N61 IQ) from plasmid pDB2536. Strain A has the following characteristics -
• additional chromosomally integrated PDIl gene integrated at the host PDIl chromosomal location.
• the URA3 gene and bacterial DNA sequences containing the ampicillin resistance gene were also integrated into the 5. cerevisiae genome at the insertion sites for the above genes.
A control strain had none of the above insertions.
Control strain [cir°] and Strain A [cir°] were transformed to leucine prototrophy with pDB2506 (recombinant transferrin), pDB2536 (recombinant non- glycosylated transferrin (N413Q, N611Q)) or pSAC35 (control). Transformants were selected on BMMD-agar.
The relative level of transferrin secretion in BMMD shake flask culture was determined for each strain/plasmid combination by rocket Immunoelectrophoresis (RIE). Figure 5 shows that both strains secreted both the glycosylated and non- glycosylated recombinant transferrins into the culture supernatant.
The levels of both the glycosylated and non-glycosylated transferrins secreted from Strain A [pDB2506] and Strain A [pDB2536] respectively, appeared higher than the levels secreted from the control strain. Hence, at least in shake flask culture, PDIl integrated into the host genome at the PDIl locus in Strain A has enhanced transferrin secretion.
Furthermore, the increase in transferrin secretion observed between control strain [pDB2536] and Strain A [pDB2536] appeared' to be at least a 100% increase by RIE. In contrast, the increase in rHA monomer secretion between control strain [pDB2305] and Strain A [pDB2305] was approximately 20% (data not shown). Therefore, the increase in transferrin secretion due to the additional copy of PDIl in Strain A was surprising large considering that transferrin has 19 disulphide bonds, compared to rHA with 17 disulphide bonds. Additional copies of the PDIl gene may be particularly beneficial for the secretion from S. cerevisiae of proteins from the transferrin family, and their derivatives.
The levels of transferrin secreted from Strain A [pDB2536] and Strain A [pDB2506] were compared by RIE for transformants grown in BMMD and YEPD (Figure 6). Results indicated that a greater than 2-fold increase in titres of both non-glycosylated recombinant transferrin (N413Q, N611Q) and glycosylated recombinant transferrin was achieved by growth in YEPD (10-20 mg.L"1 serum transferrin equivalent) compared to BMMD (2-5 mg.L"1 serum transferrin equivalent). The increase in both glycosylated and non-glycosylated transferrin titre observed in YEPD suggested that both transferrin expression plasmids were sufficiently stable under non-selective growth conditions to allow the expected increased biomass which usually results from growth in YEPD .to be translated into increased glycosylated and non-glycosylated transferrin productivity.
SDS-PAGE analysis of non-glycosylated transferrin (N413Q, N611Q) secreted from Strain A [pDB2536] and glycosylated transferrin from Strain A [pDB2506] grown in BMMD shake flask culture is shown in Figure 7. Strain A [pDB2536] samples clearly showed an additional protein band compared to the Strain A
[pSAC35] control. This extra band migrated at the expected position for the recombinant transferrin (N413Q, N611 Q) secreted from control strain [pDB2536], Strain A [pDB2506] culture supernatants appeared to contain a diffuse protein band at the position expected for transferrin. This suggested that the secreted recombinant transferrin was heterogeneous, possibly due to hyper-mannosylation at Asp413 and/or Asp61 1.
EXAMPLE 4
Comparing transferrin secretion from S. cerevisiae control strain containing pDB2711 with transferrin secretion from S. cerevisiae Strain A
Plasmid pDB2711 is as described above. Plasmid pDB2712 (Figure 22 of WO 2005/061718) was also produced with the Notl cassette in the opposite direction to pDB2711.
Control strain S. cerevisiae [cir°] was transformed to leucine prototrophy with pDB2711 and pDB2712. Transformants were selected on BMMD-agar and cryopreserved trehalose stocks of control strain [pDB2711] were prepared.
Secretion of recombinant transferrin (N413Q, N611Q) by control strain [ρDB2711], control strain [pDB2712], Strain A [pDB2536], control strain [pDB2536] and an alternative control strain [pDB2536] was compared in both BMMD and YEPD shake flask culture. PJE indicated that a significant increase in recombinant transferrin secretion had been achieved from control strain [pDB2711] with multiple episomal PDIl copies, compared to Strain A [pDB2536] with two chromosomal copies of PDIl, and control strain [pDB2536] with a single chromosomal copy of PDIl gene (Figure 8). Control strain [pDB2711] and control strain [pDB2712] appeared to secrete similar levels of rTf (N413Q, N61 IQ) into the culture media. The levels of secretion were relatively consistent between control strain [pDB2711] and control strain [pDB2712] transformants in both BMMD and YEPD media, suggesting that plasmid stability was sufficient for high-level transferrin secretion even under non-selective conditions. This is m contrast to the previous published data in relation to recombinant PDGF-BB and HSA where introduction of PDIl into multicopy 2μm plasmids was shown to be detrimental to the host.
Table 2: Recombinant transferrin titres from high cell density fermentations
Figure imgf000115_0001
Reducing SDS-PAGE analysis of transferrin secreted from control strain [pDB2711], control strain [pDB2712], Strain A [pDB2536], control strain
[pDB2536] and alternative control strain [pDB2536] in BMMD shake flask culture is shown in Figure 9. This shows an abundant protein band in all samples from control strain [pDB2711] and control strain [pDB2712] at the position expected for transferrin (N413Q, N611Q). The relative stain intensity of the transferrin (N413Q, N611Q) band from the different strains suggested that Strain
A [pDB2536] produced more than control strain [pDB2536] and alternative control strain [pDB2536], but that there was an even more dramatic increase in secretion from control strain [pDB2711] and control strain [pDB2712]. The increased recombinant transferrin secretion observed was concomitant with the increased PDIl copy number in these strains. This suggested that Pdilp levels were limiting transferrin secretion in control strain, Strain A and the alternative control strain, and that elevated PDIl copy number was responsible for increased transferrin secretion. Elevated PDIl copy number could increase the steady state expression level of PDIl so increasing the amount of Pdilp activity. There are a number of alternative methods by which this could be achieved without increasing the copy number of the PDIl gene, for example the steady state PDIl mRNA level could be increased by either increasing the transcription rate, say by use of a higher efficiency promoter, or by reducing the clearance rate of the PDIl mRNA. Alternatively, protein engineering could be used to enhance the specific activity or turnover number of the Pdilp protein.
In high cell density fermentations control strain [pDB2711] recombinant transferrin (N413Q, N61 IQ) production was measured at approximately 3g.L"' by both GP-HPLC analysis and SDS-PAGE analysis (Table 2). This level of production is several fold-higher than control strain, the alternative control strain or Strain A containing pDB2536. Furthermore, for the production of proteins for therapeutic use in humans, expression systems such as control strain [pDB2711] have advantages over those using Strain A, as they do not contain bacterial DNA sequences.
CONCLUSIONS
Secretion of recombinant transferrin from a multicopy expression plasmid (pDB2536) was investigated in S. cerevisiae strains containing an additional copy of the PDIl gene integrated into the yeast genome. Transferrin secretion was also investigated in S. cerevisiae transformed with a multicopy expression plasmid, in which the PDIl gene has been inserted into the multicopy episomal transferrin expression plasmid (pDB2711). A 5. cerevisiae strain with an additional copy of the PDIl gene integrated into the genome at the endogenous PDIl locus, secreted recombinant transferrin and non- glycosylated recombinant transferrin (N413Q. N611Q) at an elevated level compared to strains containing a single copy of PDIl . A further increase in PDIl copy number was achieved by using pDB271 1 In high cell density fermentation of the strain transformed with pDB271 1, recombinant transferrin (N413Q, N611Q) was secreted at approximately 3g.L"]. as measured by SDS-PAGE and GP-HPLC analysis. Therefore, increased PDIl gene copy number has produced a large increase in the quantity of recombinant transferrins secreted from S. cerevisiae.
The following conclusions are drawn -
1. In shake flask analysis of recombinant transferrin expression from pDB2536 (non-glycosylated transferrin (N413Q, N61 1Q) and pDB2506 (glycosylated transferrin) the S. cerevisiae strain Strain A secreted higher levels of both recombinant transferrins into the culture supernatant than control strains. This was attributed to the extra copy of PDIl integrated at the PDIl locus.
2. Control strain [pDB2711], which contained,the PDIl gene on the multicopy expression plasmid, produced a several-fold increase in recombinant transferrin (N413Q, N611Q) secretion compared to Strain A [pDB2536] in both shake flask culture and high cell density fermentation.
3. Elevated PDIl copy number in yeast such as S. cerevisiae will be advantageous during the production of desired proteins (such as a desired heterologous proteins), such as those from the transferrin family. 4. pSAC35-based plasmids containing additional copies of PDIl gene have advantages for the production of proteins from the transferrin family, and their derivatives, such as fusions, mutants, domains and truncated forms.
EXAMPLE 5
Insertion of a PDIl gene into a 2μm-like plasmid increased secretion of recombinant transferrin from various different S. cerevisiae strains
The S. cerevisiae strain JRYl 88 cir+ (National Collection of Yeast Cultures) and MT302/28B cir+ (Finnis et al, 1993, Eur. J. Biochem., 212, 201-210) was cured of the native 2μm plasmid by galactose induced over-expression of FLP from Yeρ351 -GAL-FLPl, as described by Rose and Broach (1990, Meth. EnzymoL, 185, 234-279) to create the S. cerevisiae strains JRYl 88 cir0 and MT302/28B cir°, respectively.
The S. cerevisiae strains JRYl 88 cir0, MT302/28B cir0, S150-2B cir0 (Cashmore et al, 1986, MoI Gen. Genet., 203, 154-162), CBl 1-63 cir0 (Zealey et al, 1988, MoI Gen. Genet., 211, 155-159) were all transformed to leucine prototrophy with pDB2931 (Figure 14 of WO 2005/061718) and pDB2929 (Figure 12 of WO 2005/061718). Transformants were selected on appropriately supplemented minimal media lacking leucine. Transformants of each strain were inoculated into 1OmL YEPD in 5OmL shake flasks and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Culture supematants were harvested and the recombinant transferrin titres compared by rocket immunoelectrophoresis (Figure 10). The results indicated that the transferrin titres in supematants from all the yeast strains were higher when PDIl was present in the 2μm plasmid (pDB2929) than when it was not (pDB2931) EXAMPLE 6
The construction of expression vectors containing various PDIJ genes and the expression cassettes for various heterologous proteins on the same 2μm-like plasmid
PCR amplification and cloning of PDIl genes into Ylpladll:
The PDIl genes from S. cerevisiae S288c and S. cerevisiae SKQ2n were amplified by PCR to produce DNA fragments with different lengths of the 5'- untranslated region containing the promoter sequence. PCR primers were designed to permit cloning of the PCR products into the £coRI and BamHl sites of YIplac211 (Gietz & Sugino, 1988, Gene, 74, 527-534). Additional restriction endonuclease sites were also incorporated into PCR primers to facilitate subsequent cloning. Table 3 describes the plasmids constructed and Table 4 gives the PCR primer sequences used to amplify the PDIl genes. Differences in the PDIl promoter length within these YIplac211 -based plasmids are described in Table 3.
pDB2939 (Figure 27 of WO 2005/061718) was produced by PCR amplification of the PDIl gene from 5. cerevisiae S288c genomic DNA with oligonucleotide primers DS248 and DS250 (Table 5), followed by digesting the PCR product with EcoKL and BamΗl and cloning the approximately 1.98-kb fragment into YIplac211 (Gietz & Sugino, 1988, Gene, 74, 527-534), that had been cut with £cøRI and BaniHl. DNA sequencing of pDB2939 identified a missing 'G' from within the DS248 sequence, which is marked in bold in Table 4. Oligonucleotide primers used for sequencing the PDIl gene are listed in Table 5, and were designed from the published S288c PDIl gene sequence (PD11/YCL043C on chromosome III from coordinates 50221 to 48653 plus 1000 base pairs of upstream sequence and 1000 base pairs of downstream sequence. (http://wwvy.veastgenorne.org/ Genebank Accession number NCOOl 135).
Table 3: YIplac21 1 -based Plasmids Containing PDIl Genes
Figure imgf000120_0001
Table 4: Oligonucleotide Primers for PCR Amplification of S cerevisiae PDIl Genes
Figure imgf000121_0001
Table 5: Oligonucleotide Primers for DNA Sequencing S. cerevisiae PDIl Genes
Figure imgf000122_0001
Plasmids pDB2941 (Figure 28 of WO 2005/061718) and pDB2942 (Figure 29 of WO 2005/061718) were constructed similarly using the PCR primers described in Tables 3 and 4, and by cloning the approximately 1.90-kb and 1.85-kb EcoRl- BamEl fragments, respectively, into YIplac211. The correct DNA sequences were confirmed for the PDU genes in pDB2941 and pDB2942. The S. cerevisiae SKQ2n PDIl gene sequence was PCR amplified from plasmid DNA containing the PDIl gene from pMA3a:C7 (US 6:291,205); also known as Clone C7 (Crouzet & Tuite, 1987, supra; Farquhar et al, 1991. supra). The SKQ2n PDU gene was amplified using oligonucleotide primers DS248 and DS250 (Tables 3 and 4). The approximately 2.01-kb PCR product was digested with EcoRl and BamHl and ligated into YIplac21 1 (Gietz & Sugino, 1988, Gene, 74, 527-534) that has been cut with EcoKL and BamHl, to produce plasmid pDB2943 (Figure 30 of WO 2005/061718). The 51 end of the SKQ2n PDIl sequence is analogous to a blunt-ended Spel-ύte extended to include the EcoBl, Sad, SnaBl, Pad, Fsel, Sfil and Smal sites, the 3' end extends up to a site analogous to a blunt-ended Bsu36l site, extended to include a Smal, SnaBl and BamHl sites. The PDIl promoter length is approximately 210bp. The entire DNA sequence was determined for the PDIl fragment using oligonucleotide primers given in Table 5. This confirmed the presence of a coding sequence for the PDI protein of S. cerevisiae strain SKQ2n (NCBI accession number CAA38402), but with a serine residue at position 114 (not an arginine residue as previously published). Similarly, in the same way as in the S. cerevisiae S288c sequence in pDB2939, pDB2943 also had a missing 'G' from within the DS248 sequence, which is marked in bold in Table 4.
Plasmids pDB2963 (Figure 31 of WO 2005/061718) and pDB2945 (Figure 32 of WO 2005/061718) were constructed similarly using the PCR primers described in Tables 3 and 4, and by cloning the approximately 1.94-kb and 1.87-kb EcoRI- BamHl fragments, respectively, into YIplac211. The expected DNA sequences were confirmed for the PDIl genes in pDB2963 and pDB2945, with a serine codon at the position of amino acid 114. The construction of pSAC35-based rHA expression plasmids with different PDIl genes inserted at the A'cml-site after REP2:
pSAC35-based plasmids were constructed for the co-expression of rHA with different PDIl genes (Table 6).
Table 6: pSAC35-based plasmids for co-expression of rHA with different PDIl genes
Figure imgf000124_0001
.The rHA expression cassette from pDB2243 (Figure 33 of WO 2005/061718, as also described in WO 00/44772) was first isolated on a 2,992-bp Notl fragment, which subsequently was cloned into the Nofl-site of pDB2688 (Figure 4 of WO 2005/061718) to produce pDB2693 (Figure 34 of WO 2005/061718). pDB2693 was digested with SnaBl, treated with calf intestinal alkaline phosphatase, and ligated with SnciBl fragments containing the PDIl genes from pDB2943, ρDB2963, pDB2945; pDB2939, pDB2941 and pDB2942. This produced plasmids pDB2976 to pDB2987 (Figures 35 to 46 of WO 2005/061718). PDIl transcribed in the same orientation as REP2 was designated "orientation A", whereas PDIl transcribed in opposite orientation to REP2 was designated "orientation B" (Table 6).
The construction of pSAC35-based transferrin expression plasmids with different PDIl genes inserted at the Xcml-site after REP2:
pSAC35-based plasmids were constructed for the co-expression of recombinant transferrin (N413Q, N61 IQ) with different PDIJ genes (Table 7).
Table 7: pSAC35-based plasmids for co-expression of transferrin with different PDIl genes
Figure imgf000125_0001
In order to achieve this, the Notl expression cassettes for rHA expression were first deleted from pDB2976, pDB2978, and pDB2980 by Notl digestion and circularisation of the vector backbone. This produced plasmids pDB3081 (Figure 47 of WO 2005/061718), pDB3083 (Figure 48 of WO 2005/061718) and pDB3084 (Figure 49 of WO 2005/061718) as described in Table 8.
Table 8: pSAC35-based plasmids with different PDIl genes
Figure imgf000126_0001
The 3;256-bp Notl fragment from pDB2928 (Figure 11 of WO 2005/061718) was cloned into the Notl-sites of pDB3081, pDB3083 and pDB3084, such that transcription from the transferrin gene was in the same direction as LEU2. This produced plasmids pDB3085 (Figure 50 of WO 2005/061718), pDB3086 (Figure 51 of WO 2005/061718) and pDB3087 (Figure 52 of WO 2005/061718) as described in Table 7.
EXAMPLE 7
Insertion and optimisation of a PDIl gene in the 2μm-like plasmid increased the secretion of recombinant human serum albumin by various different S. cerevisiae strains
The S. cerevisiae strains JRYl 88 cir°, MT302/28B cir°, S150-2B cir0 , CBl 1-63 cϊr° (all described above), AH22 cir0 (Mead et ai, 1986, MoI. Gen. Genet., 205, 417-421) and DS569 cir0 (Sleep et al., 1991, Bio/Technology, 9.. 183-187) were transformed to leucine prototrophy with either pDB2244 (WO 00/44772), pDB2976 (Figure 35 of WO 2005/061718), pDB2978 (Figure 37 of WO 2005/061718) or pDB2980 (Figure 39 of WO 2005/061718) using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I. protocol 2; (Ito et al, 1983, J. BacterioL, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD-agar plates with appropriate supplements, and were subsequently patched out on BMMD-agar plates with appropriate supplements.
Transformants of each strain were inoculated into 1OmL YEPD in 5OmL shake flasks and incubated in an orbital shaker at 300C, 200rρm for 4-days. Culture supernatants were harvested and the recombinant albumin titres compared by rocket Immunoelectrophoresis (Figures 11 and 12). The results indicated that the albumin titres in the culture supernatants from all the yeast strains were higher when PDIl was present in the 2μm plasmid than when it was not (pDB2244). The albumin titre in the culture supernatants in the absence of PDIl on the plasmid was dependant upon which yeast strain was selected as the expression host, however, in most examples tested the largest increase in expression was observed when PDIl with the long promoter (~210-bp) was present in the 2μm plasmid (pDB2976). Modifying the PDIl promoter by shortening, for example to delete regulation sites, had the affect of controlling the improvement. For one yeast strain, known to be a high rHA producing strain (DS569) a shorter promoter was preferred for optimal expression.
EXAMPLE 8
PDIl on the 2μm-based plasmid enhanced the secretion of recombinant alb umin fusions.
The affect of co-expression of the S. cerevisiae SKQ2n PDIl gene with the long promoter (~210-bp) upon the expression of recombinant albumin fusions was investigated.
The plasmids defined in Table 9 below were generated as described in Example 9 of WO 2005/061718, the contents of which are incorporated herein by reference.
Table 9: Summary of plasmids encoding albumin fusion proteins
Albumin fusion Plasmid name
With PDU Without PDU endostatin-albumin pDB3100 PDB3099 angiostatin-albumin pDB3107 pDB2765
Kringle5-(GGS)4GG-albumin pDB3104 ρDB2773
DX-890-(GGS)4GG-albumin pDB3102 pDB3101
DPI-14-(GGS)4GG-albumin pDB3103 pDB2679
Axokine™-(GGS)4GG-albumin pDB3106 pDB2618 human IL10-(GGS)4-GG-albumin pDB3105 pDB2621
The same control yeast strain as used in previous examples was transformed to leucine prototrophy using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2; (Ito et al, 1983, J. Bacteviol, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD- agar plates, and were subsequently patched out on BMMD-agar plates. Cryopreserved trehalose stocks were prepared from 1OmL BMMD shake flask cultures (24 hrs, 3O°C, 200rpm).
Transformants of each strain were inoculated into 1OmL BMMD in 5OmL shake flasks and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Culture supernatants were harvested and the recombinant albumin fusion titres compared by rocket Immunoelectrophoresis (Figure 13). The results indicated that the albumin fusion titre in the culture supernatants from yeast strain was higher when PDIl was present in the 2μm plasmid than when it was not.
The increase in expression of the albumin fusions detected by rocket Immunoelectrophoresis was further studied by SDS-PAGE analysis. BMMD shake flask cultures of YBX7 expressing various albumin-fusions were grown for 4-days in an orbital shaker at 3O°C, 200rpm. A sample of the culture supernatant was analysed by SDS-PAGE (Figure 14). A protein band of the expected size for the albumin fusion under study was observed increase in abundance.
EXAMPLE 9
Co-expression of S. cerevisiae 0RM2 and recombinant transferrin on a 2μm- based plasmid
The S". cerevisiae Control Strain and Strain A (as described in Example 3) were selected to investigate the effect on transferrin secretion when the transferrin and 0RM2 genes were co-expressed from the 2 μm -based plasmids. The Control Strain and Strain A were transformed to leucine prototrophy by plasmids pDB3090, pDB3092 and pBD3093 (containing expression cassettes for rTf (N413Q, N611Q) and for 0RM2), as well as a control plasmid pDB2931 (containing the rTf (N413Q, N611Q) expression cassette without 0RM2). The construction of these plasmids is described in Example 10 of WO 2005/061718, the contents of which are incorporated herein by reference. Transformants were selected on BMMD agar and patched out on BMMD agar for subsequent analysis.
To investigate the effect of 0RM2 co-expression on transferrin secretion, 1 OmL selective (BMMD) and non-selective (YEPD) liquid media were inoculated with strains containing the ORM2/transferrin co-expression plasmids. The shake flask culture was then incubated at 3O°C with shaking (200 rpm) for 4 days. The relative level of transferrin secretion was determined by rocket gel Immunoelectrophoresis (RIE) (Figure 15).
Levels of transferrin secreted from Control Strain [pDB3090] and Control Strain [pDB3092] were greater than the levels from Control Strain [pDB2931] in both BMMD and YEPS media. Similarly, the levels of transferrin secreted from both Strain A [pDB3090] and Strain A [pDB3093] were greater than the levels from Strain A [pDB2931] in both BMMD and YEPS media. Transferrin secretion from all Strain A transformants was higher than the Control Strain transformants grown in the same media. Strain A contains an additional copy of PDIl in the genome, which enhanced transferrin secretion. Therefore in Strain A, the increased expression of 0RM2 and PDIl had a cumulative effect on the secretion of transferrin.
EXAMPLE 10
Co-expression of S. cerevisiae PSEl and recombinant transferrin on a 2μm- based plasmid
The S. cerevisiae Control Strain was transformed to leucine prototrophy by plasmids, pDB3097 and pBD3098 (containing expression cassettes for rTf
(N413Q, N61 1Q) and for PSEl), as well as a control plasmid pDB2931 (containing the rTf (N413Q, N611Q) expression cassette without PSEl). The construction of these plasmids are described in Example 11 of WO 2005/061718. the contents of which are incorporated herein by reference. Transformants were selected on BMMD agar and patched out on BMMD agar for subsequent analysis.
To investigate the effect of PSEl expression on transferrin secretion, flasks containing 1OmL selective (BMMD) liquid media were inoculated with strains containing the PSEl /transferrin co-expression plasmids. The shake flask culture was then incubated at 3O°C with shaking (200 rpm) for 4 days. The relative level of transferrin secretion was determined by rocket gel Immunoelectrophoresis (RIE) (Figure 16).
Levels of transferrin secreted from Control Strain [pDB3097] and Control Strain [pDB3098] were greater than the levels from Control Strain [pDB2931] in BMMD media. Therefore, expression of PSEl from the 2μm-based plasmids had enhanced transferrin secretion from S. cerevisiae. Transferrin secretion was improved with the PSEl gene transcribed in either direction relative to the REP2 gene in pDB3097 and pDB3098.
EXAMPLE 11
Co-expression of S. cerevisiae SSAl and recombinant transferrin on a 2μm- based plasmid
The S. cerevisiae Control Strain was transformed to leucine prototrophy by plasmids, pDB3094 and pBD3095 (containing expression cassettes for rTf (N413Q, N61 1Q) and for SSAl), as well as a control plasmid pDB2931 (containing the rTf (N413Q, N61 1 Q) expression cassette without SSAl). The construction of these plasmids are described in Example 12 of WO 2005/061718, the contents of which are incorporated herein by reference. Transformants were selected on BMMD agar and patched out on BMMD agar for subsequent analysis. To investigate the effect of SSAl expression on transferrin secretion, flasks containing 1OmL selective (BMMD) liquid media were inoculated with strains containing the S SAi /transferrin co-expression plasmids. The shake flask cultures were incubated at 3O°C with shaking (200 rpm) for 4 days. The relative level of transferrin secretion was determined by rocket gel Immunoelectrophoresis (RIE) (Figure 17).
Levels of transferrin secreted from Control Strain [pDB3095] were greater than the levels from Control Strain [pDB2931] and Control Strain [pDB3094] in BMMD media. Therefore, expression of SSAl from the 2μm-based plasmids had enhanced transferrin secretion from S. cerevisiae. Transferrin secretion was improved with the SSAl gene transcribed in the opposite direction relative to the REP2 gene in pDB3094.
EXAMPLE 12
PDIl gene disruption, combined with a PDIl gene on the 2μm-based plasmid enhanced the secretion of recombinant albumin and plasmid stability.
Single stranded oligonucleotide DNA primers listed in Table 11 were designed to amplify a region upstream of the yeast PDIl coding region and another a region downstream of the yeast PDIl coding region.
Table 11 : Oligonucleotide primers
Figure imgf000133_0001
Primers DS299 and DS300 amplified the 5' region of PDIl by PCR, while primers DS301 and DS302 amplified a region 3' of PDIl, using genomic DNA derived S288c as a template. The PCR conditions were as follows: lμL S288c template DNA (at O.Olng/μL, O.lng/μL, lng/μL, lOng/μL and lOOng/μL), 5μL lOXBuffer (Fast Start Taq+Mg, (Roche)), lμL 1OmM dNTP's, 5μL each primer (2μM), 0.4μL Fast Start Taq, made up to 50μL with H2O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9700. The conditions were: denature at 95°C for 4min [HOLD], then [CYCLE] denature at 95°C for 30 seconds, anneal at 45°C for 30 seconds, extend at 72°C for 45 seconds for 20 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C. The 0.22kbp PDIl 5' PCR product was cut with Notl and HmdIIL while the 0.34kbp PDIl 3' PCR product was cut with HmdIII and P stl.
Plasmid pMCS5 (HoheiseL 1994, Biotechniques 17, 456-460) (Figure 85 of WO 2005/061718) was digested to completion with Hinάlll, blunt ended with T4 DNA polymerase plus dNTPs and religated to create pDB2964 (Figure 86 of WO 2005/061718).
Plasmid pDB2964 was Hindlϊi digested, treated with calf intestinal phosphatase, and ligated with the 0.22kbp PDIl 5' PCR product digested with Notl and HindHl and the 0.34kbp PDIl 3' PCR product digested with Hinάlll and PsA to create pDB3069 (Figure 87 of WO 2005/061718) which was sequenced with forward and reverse universal primers and the DNA sequencing primers DS303, DS304, DS305 and DS306 (Table 11).
Primers DS234 and DS235 (Table 12) were used to amplify the modified TRPl marker gene from Ylplac204 (Gietz & Sugino, 1988, Gene, 74, 527-534), incorporating Hindlϊl restriction sites at either end of the PCR product. The PCR conditions were as follows: l μL template YIplac204 (at O.Olng/μL, O.lng/μL, lng/μL, lOng/μL and lOOng/μL), 5μL lOXBuffer (Fast Start Taq+Mg, (Roche)), lμL 1OmM dNTP's, 5μL each primer (2μM), 0.4μL Fast Start Taq, made up to 50μL with H2O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9600. The conditions were: denature at 95°C for 4min [HOLD], then [CYCLE] denature at 95°C for 30 seconds, anneal for 45 seconds at 45°C, extend at 72°C for 90sec for 20 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C. The 0.86kbp PCR product was digested with Hindlll and cloned into the HmdIIl site of pMCS5 to create pDB2778 (Figure 88 of WO 2005/061718). Restriction enzyme digestions and sequencing with universal forward and reverse primers as well as DS236, DS237, DS238 and DS239 (Table 12) confirmed that the sequence of the modified TRPl gene was correct. Table 12: Oligonucleotide primers
Figure imgf000135_0001
The 0.86kbp TRPl gene was isolated from pDB2778 by digestion with Hinάlll and cloned into the Hindlll site of pDB3069 to create pDB3078 (Figure 89 of WO 2005/061718) and pDB3079 (Figure 90 of WO 2005/061718). A 1.41kb pdil :TRPl disrupting DNA fragment was isolated from pDB3078 or pDB3079 by digestion with NotllPstl. Yeast strains incorporating a TRPl deletion (trplA) were to be constructed in such a way that no homology to the TRPl marker gene (pDB2778) should left in the genome once the trplΔ had been created, so preventing homologous recombination between future TRPl containing constructs and the TRPl locus. In order to achieve the total removal of the native TRPl sequence from the genome of the chosen host strains, oligonucleotides were designed to amplify areas of the 5' UTR and 3' UTR of the TRPl gene outside of TRPl marker gene present on integrating vector YIplac204 (Gietz & Sugino, 1988, Gene, 74, 527-534). The YIplac204 TRPl marker gene differs from the native/chromosomal TRPl gene in that internal Hzndlll, Pstl and Xbal sites were removed by site directed mutagenesis (Gietz & Sugino, 1988, Gene, 74, 527-534). The YIplac204 modified TRPl marker gene was constructed from a 1.453kbp blunt-ended genomic fragment EcoRλ fragment, which contained the TRPl gene and only 102bp of the TRPl promoter (Gietz & Sugino, 1988, Gene, 74, 527-534). Although this was a relatively short promoter sequence it was clearly sufficient to complement trpl auxotrophic mutations (Gietz & Sugino, 1988, Gene, 74, 527- 534). Only DNA sequences upstream of the EcoKl site, positioned 102bp 5' to the start of the TRPl ORF were used to create the 5' TRPl UTR. The selection of the 3' UTR was less critical as long as it was outside the 3' end of the functional modified TRPl marker, which was chosen to be 85bp downstream of the translation stop codon.
Single stranded oligonucleotide DNA primers were designed and constructed to amplify the 5' UTR and 3' UTR regions of the TRPl gene so that during the PCR amplification restriction enzyme sites would be added to the ends of the PCR products to be used in later cloning steps. Primers DS230 and DS231 (Table 12) amplified the 5' region of TRPl by PCR, while primers DS232 and DS233 (Table 12) amplified a region 3' of TRPl, using S288c genomic DNA as a template. The PCR conditions were as follows: l μL template S288c genomic DNA (at O.Olng/μL, O.lng/μL, lng/μL, lOng/μL and lOOng/μL), 5μL lOXBuffer (Fast Start Taq+Mg; (Roche)), lμL 1 OmM dNTP's, 5μL each primer (2μM), 0.4μL Fast Start Taq, made up to 50μL with H2O. PCRs were performed using a Perkin- Elmer Thermal Cycler 9600. The conditions were: denature at 95°C for 4min [HOLD], then [CYCLE] denature at 95°C for 30 seconds, anneal for 45 seconds at 45°C, extend at 72°C for 90sec for 20 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C.
The 0.19kbp TRPl 5' UTR PCR product was cut with EcόRl and Hinάlll, while the 0.2kbp TRPl 3' UTR PCR product was cut with BamUl and Hindlll and ligated into pAYE505 linearised with BamHl/EcoRl to create plasmid pDB2777 (Figure 91 of WO 2005/061718). The construction of pAYE505 is described in WO 95/33833 . DNA sequencing using forward and reverse primers, designed to prime from the plasmid backbone and sequence the cloned inserts, confirmed that in both cases the cloned 5' and 3' UTR sequences of the TRPl gene had the expected DNA sequence. Plasmid pDB2777 contained a TRPl disrupting fragment that comprised a fusion of sequences derived from the 5' and 3' UTRs of TRPl. This 0.383kbp TRPl disrupting fragment was excised from pDB2777 by complete digestion with EcoRl.
Yeast strain DXYl (Kerry- Williams et al, 1998, Yeast, 14, 161-169) was transformed to leucine prototrophy with the albumin expression plasmid pDB2244 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST- 1, protocol 2; (Ito et al, 1983, J. BacterioL, 153, 163; Elble, 1992, Biotechniques, 13, 18)) to create yeast strain DXYl [pDB2244]. The construction of the albumin expression plasmid pDB2244 is described in WO 00/44772. Transformants were selected on BMMD-agar plates, and were subsequently patched out on BMMD- agar plates. Cryopreserved trehalose stocks were prepared from 1OmL BMMD shake flask cultures (24 hrs, 3O°C, 200rpm). DXYl [pDB2244] was transformed to tryptophan autotrophy with the 0.383kbp EcoRl TRPl disrupting DNA fragment from pDB2777 using a nutrient agar incorporating the counter selective tryptophan analogue. 5-fluoroanthranilic acid (5-FAA), as described by Toyn el al., (2000 Yeast 16, 553-560). Colonies resistant to the toxic effects of 5-FAA were picked and streaked onto a second round of 5-FAA plates to confirm that they really were resistant to 5-FAA and to select away from any background growth. Those colonies which grew were then were re-patched onto BMMD and BMMD plus tryptophan to identify which were tryptophan auxotrophs.
Subsequently colonies that had been shown to be tryptophan auxotrophs were selected for further analysis by transformation with YCplac22 (Gietz & Sugino, 1988, Gene, 74, 527-534) to ascertain which isolates were trpl,
PCR amplification across the TRPl locus was used to confirm that the trp" phenotype was due to a deletion in this region. Genomic DNA was prepared from isolates identified as resistant to 5-FAA and unable to grow on minimal media without the addition of tryptophan. PCR amplification of the genomic TRPl locus with primers CED005 and CED006 (Table 12) was achieved as follows: lμL template genomic DNA, 5μL lOXBuffer (Fast Start Taq+Mg, (Roche)), lμL 1OmM dNTP's, 5μL each primer (2μM), 0.4μL Fast Start Taq, made up to 50μL with H2O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9600. The conditions were: denature at 94°C for lOmin [HOLD], then [CYCLE] denature at 94°C for 30 seconds, anneal for 30 seconds at 55°C, extend at 72°C for 120sec for 40 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C. PCR amplification of the wild type TRPl locus resulted in a PCR product of 1.34kbp in size, whereas amplification across the deleted TRPl region resulted in a PCR product 0.84kbp smaller at 0.50kbp. PCR analysis identified a DXYl derived trp" strain (DXYl trpl A [pDB2244]) as having the expected deletion event. The yeast strain DXYl trplΔ [pDB2244] was cured of the expression plasmid pDB2244 as described by Sleep et ah, (1991, Bio/Technology, 9, 183-187). DXYl trplA cir0 was re-transformed the leucine prototrophy with either pDB2244, pDB2976, pDB2977, pDB2978, pDB2979, pDB2980 or pDB2981 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST- I5 protocol 2; (Ito et al, 1983, J Bacteriol, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD-agar plates supplemented with tryptophan, and were subsequently patched out on BMMD-agar plates supplemented with tryptophan. Cryopreserved trehalose stocks were prepared from 1OmL BMMD shake flask cultures supplemented with tryptophan (24 hrs, 3O°C, 200rpm).
The yeast strains DXYl trplΔ [pDB2976], DXYl trplΔ [pDB2977], DXYl trplA [pDB2978], DXYl trplΔ [pDB2979], DXYl trplΔ [pDB2980] or DXYl trplΔ [pDB2981] was transformed to tryptophan prototrophy using the modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2;
(Ito et al, 1983, J Bacteriol., 153, 163; Elble, 1992, Biotechniques, 13, 18)) with a
1.41kb pdilr. TRPl disrupting DNA fragment was isolated from ρDB3078 by digestion with NotVPstl. Transformants were selected on BMMD-agar plates and were subsequently patched out on BMMD-agar plates.
Six transformants of each strain were inoculated into 1 OmL YEPD in 5OmL shake flasks and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Culture supernatants and cell biomass were harvested. Genomic DNA was prepared (Lee, 1992, Biotechniques, 12, 677) from the tryptophan prototrophs and DXYl [pDB2244]. The genomic PDIl locus amplified by PCR of with primers DS236 and DS303 (Table 11 and 12) was achieved as follows: l μL template genomic DNA, 5μL lOXBuffer (Fast Start Taq+Mg, (Roche)), lμL 1OmM dNTP's, 5μL each primer (2μM), 0.4μL Fast Start Taq, made up to 50μL with H2O. PCRs were performed using a Perkin-Elmer Thermal Cycler 9700. The conditions were: denature at 94°C for 4min [HOLD], then [CYCLE] denature at 94°C for 30 seconds, anneal for 30 seconds at 50°C, extend at 72°C for 60sec for 30 cycles, then [HOLD] 72°C for lOmin and then [HOLD] 4°C. PCR amplification of the wild type PDIl locus resulted in no PCR product, whereas amplification across the deleted PDIl region resulted in a PCR product 0.65kbp. PCR analysis identified that all 36 potential pdil::TRPl strains tested had the expected pdilr. TRPl deletion.
The recombinant albumin titres were compared by rocket Immunoelectrophoresis (Figure 18). Within each group, all six pdil::TRPl disruptants of DXYl trplΛ [pDB2976], DXYl trplΔ [pDB2978], DXYl trplΛ [pDB2980], DXYl trplΔ [pDB2977] and DXYl trplΔ [pDB2979] had very similar rHA productivities. Only the ύxpdil::TRPl disruptants of DXYl trplΔ [pDB2981] showed variation in rHA expression titre. The six pdilr. TRPl disruptants indicated in Figure 18 were spread onto YEPD agar to isolate single colonies and then re-patched onto BMMD agar.
Three single celled isolates of DXYl trplΔ pdilr. TRPl [pDB2976], DXYl trplΔ pdilr. TRPl [pDB2978], DXYl trplΔ pdilr. TRPl [pDB2980], DXYl trplΔ pdilr. TRPl [pDB2977], DXYl trplΔ pdilr. TRPl [pDB2979] and DXYl trplΔ pdilr. TRPl [pDB2981] along with DXYl [pDB2244], DXYl [pDB2976], DXYl
[pDB2978], DXYl [pDB2980], DXYl [pDB2977], DXYl [pDB2979] and DXYl
[pDB2981] were inoculated into 1OmL YEPD in 5OmL shake flasks and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Culture supernatants were harvested and the recombinant albumin titres were compared by rocket
Immunoelectrophoresis (Figure 19). The thirteen wild type PDIl andpdil rTRPl disruptants indicated in Figure 19 were spread onto YEPD agar to isolate single colonies. One hundred single celled colonies from each strain were then re- patched onto BMMD agar or YEPD agar containing a goat anti-HSA antibody to ' detect expression of recombinant albumin (Sleep et ah, 199L Bio/Technolog)', 9. 183-187) and the Leu+/rHA+, Leu+/rHA-. Leu-/rHA+ or Leu-/rHA- phenotype of each colony scored (Table 13).
Table 13:
Figure imgf000141_0001
These data indicate plasmid retention is increased when the PDIl gene is used as a selectable marker on a plasmid in a host strain having no chromosomally encoded PDI, even in a non-selective medium such as the exemplified rich medium.
Example 13
Construction of the pSAC35-based expression vectors pDB3175-pDB3182 for co-expression of PDIl with recombinant human albumin or transferrin (N413O, N6110)fwm the SnaBI/Notl-site in the 2μm UL-region
The Notl expression cassette from the pSAC35-based expression vector, pAYE316 (Sleep et al, 1991, Biotechnology (N Y), 9, 183-187), designed for the secretion of recombinant human albumin, was cloned into the unique NotI-site of the E. coli cloning vector ρBST(+) (Sleep et al, 2001, Yeast, 18, 403-421) to produce plasmid pQC262e. pQC262e was subsequently modified by site-directed mutagenesis (Kunkel et al, 1987, Methods Enzymol, 154, 367-382) with oligonucleotide LRE49 (Table 14) to introduce a unique Asp718I-site immediately upstream of the NotI-site at the ADH1 terminator region of the expression cassette.
Table 14: Mutagenic Oligonucleotide
Figure imgf000142_0001
This produced plasmid pAYE560 (Figure 21). The S. cerevisiae SKQ2n PDIl gene with the long (~210-bp) promoter was isolated on 1.96-kb Smal fragment from pDB2952 (Figure 46 in WO 2005/061719, the contents of which are incorporated herein by reference). The pDB2952 PDIl fragment was cloned into the unique Asp718I-site of pAYE560, following digestion with Asp718I, filling the 3 '-recessed ends using T4 DNA polymerase, and calf intestinal alkaline phosphatase treatment of the blunt-ended product. This produced plasmids pDB3171 (Figure 22) with the PDIl gene transcribed in the same direction as rHA, and pDB3172 (Figure 23) with converging transcription of the PDIl and rHA genes.
pDB3175 and pDB3176 (Figures 24 and 25) were produced by cloning the ~ 5.1- kb rHA→PZ>/7→ Noll expression cassette from pDB3171 into pSAC35. which had been digested with Notl and calf intestinal alkaline phosphatase. pDB3177 and pDB3178 (Figures 26 and 27) were produced similarly by cloning the ~ 5.1- kb Noll rHA— ><— PDIl expression cassette from pDB3172 into pSAC35 digested with Noll and calf intestinal alkaline phosphatase.
To construct Noll expression cassettes for co-expression of recombinant unglycosylated human transferrin (N413Q, N611Q) and the S cerevisiae SKQ2n PDIl gene with the long (~210-bp) promoter, the ~ 6.1-kb Aflϊl-Sphl fragment from pDB3171 was ligated with the ~ 2.4-kb Aflll-Sphl fragment from pDB2928 (Figure 11 of WO 2005/061718, the contents of which are incorporated herein by reference), thus replacing the rHA coding and adjacent sequences with those for transferrin (N413Q, N61 1 Q) secretion. This produced plasmids pDB3173 (Figure 28) with the PDIl gene transcribed in the same direction as rTf (N413 Q, N611 Q), and pDB3174 (Figure 29) with converging transcription of the PDIl and rTf (N413Q, N61 IQ) genes.
pDB3179 and pDB3180 (Figures 30 and 31) were produced by cloning the ~ 5.2- kb rTf→ PDIl → Notl expression cassette from pDB3173 into pSAC35, which had been digested with Notl and calf intestinal alkaline phosphatase. pDB3181 and pDB3182 (Figures 32 and 33) were produced by cloning the ~ 5.2-kb Notl rTf→<— PDIl expression cassette from pDB3174 into pSAC35 digested with Notl and calf intestinal alkaline phosphatase. Example 14
S. cerevisiae PDIl at the SnaBI/Notl-site in the UL-region of pSAC35-based expression vectors as a selectable marker in S. cerevisiae strain DXYl Δtrpl pdil::TRPl
The yeast strains DXYl (Kerry-Williams el a!., 1998, Yeast, 14, 161-169) and DXYl Λtrpl (see Example 13 of WO 2005/061718, the contents of which are incorporated herein by reference) were transformed to leucine prototrophy with pSAC35-based plasmids pAYE316, pDB3175, pDB3176, pDB3177, pDB3178, pDB2931, pDB3179, pDB3180, pDB3181 and pDB3182 using a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I , protocol 2; (Ito et al, 1983, J Bacteriol, 153, 163; Elble, 1992, Biotechniques, 13, 18)). Transformants were selected on BMMD-agar plates with appropriate supplements, and were subsequently patched out on BMMD-agar plates with appropriate supplements.
DXYl Δtrpl [pDB3175], DXYl Δtrpl [pDB3176], DXYl Atrpl [pDB3177], DXYl Atrpl [pDB3178], DXYl Atrpl [pDB3179], DXYl Atrpl [pDB3180], DXYl Atrpl [pDB3181], and DXYl Atrpl [pDB3182] were transformed to tryptophan prototrophy using the modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2; (Ito el al, 1983, J Bacteriol., 153, 163; Elble, 1992, Biotechniques, 13, 18)) with a 1.41-kb pdil: TRPl disrupting DNA fragment isolated from pDB3078 by digestion with NotllPstl. (see Example 13 of WO 2005/061718). Transformants were selected on BMMD-agar plates and were subsequently patched out on BMMD-agar plates.
Disruption of the PDIl gene with the TRPl marker was confirmed by diagnostic PCR amplification of an approximately 810-bp product using oligonucleotide primers CF247 and DS236 (Table 15). CF247 binds in the PDIl promoter region upstream of the disruption site and DS236 binds within the TRPl gene. Table 15: Oligonucleotide Sequencing Primers
Figure imgf000145_0001
DXYl Δtrpl, DXYl Atrpl containing pDB3175-pDB3182 and putative pdil:: TRPl -disruptants were inoculated into 1 OmL YEPD in 5OmL shake flasks, and incubated in an orbital shaker at 3O°C, 200rpm for 4-days. Genomic DNA was prepared (Lee, 1992, Biotechniques, 12, 677) from the biomass for subsequent use as template DNA in diagnostic PCR.
0.5 μL template genomic DNA, 2.5 μL lOxBuffer (Fast Start Taq+Mg, (Roche)), 0.5μL 1OmM dNTP's, 2.5μL each primer (2μM), 0.2μL Fast Start Taq were mixed and made up to 25 μL with H2O. PCRs were performed using a Dyad™ DNA Engine Peltier thermal cycler (GRI) as follows; denaturation at 95°C for 4 mins, then 25 cycles of 95°C for 30s, 55°C for 30s and 72°C for 1 min, followed by extension at 72°C for 10 mins. A PCR product of ~810-bp was only amplified from DNA containing the TRPl gene integrated at the pdil locus. DXYl Atrpl pdil::TRPl containing pDB3175-pDB3182 were successfully identified. As expected, no PCR product was generated for the controls DXYl Atrpl or DXYl Atrpl containing pDB3175-pDB3182.
DXYl containing pDB3175-pDB3182 were compared with DXYl Atrpl pdil::TRPl containing pDB3175-pDB3182 for plasmid stability and secretion of recombinant human albumin or transferrin (N413Q. N61 IQ) in YEPD shake flask culture. 1OmL YEPD shake flasks were inoculated with the above strains and grown for 4-days at 30°C, 200rpm. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 3O°C. Plasmid stability was scored as the percentage of colonies able to grow on both media. The results shown in Tables 16 and 17 demonstrated that all of the plasmids pDB3175- pDB3182 were less than 100% stable in DXYl, regardless of the relative orientations of the PDIl and rTf/rHA genes cloned at the SnaBVNotl-site. However, 100% plasmid stability was determined in all cases in DXYl Atrpl pdil. :TRPl containing pDB3175-pDB3182 . Hence, use of PDIl as the sole selectable marker at the SnaBl/Notl-site of pSAC35-based vectors resulted in 100% plasmid stability in rich media.
Table 16: Plasmid stability of pSAC35-based vectors containing a recombinant albumin gene and the S cerevisiae PDIl gene at the SnaBI/Notl-site in the UL- region in strains DXYl and DXYl Δtrpl pdilr. TRPl. 1OmL YEPD shake flasks were inoculated with DXYl [pDB3175], DXYl [pDB3176], DXYl [pDB3177], DXYl [pDB3178], DXYl Atrpl pdilr. TRPl [pDB3175], DXYl Atrpl pdil::TRPl [pDB3176], DXYl Δirpl pdilr. TRPl [pDB3177], and DXYl Atrpl pdilr. TRPl [pDB3178] were grown for 4-days at 30°C; 200φm. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 30°C. Plasmid stability was scored as the percentage of colonies able to grow on both media.
Figure imgf000147_0001
* Arrows indicate the direction of transcription relative to the LEU2 gene. Table 17: Plasmid stability of pSAC35-based vectors containing a recombinant transferrin gene and the S. cerevisiae PDIl gene at the SnαBLWo/I-site in the UL- region in strains DXYl and DXYl Δtrpl pdil::TRPl. 1 OmL YEPD shake flasks were inoculated with DXYl [pDB3179], DXYl [pDB3180], DXYl [pDB3181], DXYl [pDB3182]: DXYl Atrpl pdilr. TRPl [pDB3179], DXYl Δtrpl pdil::TRPl [pDB3180], DXYl Δtrpl pdil::TRPl [pDB3181]: and DXYl Atrpl pdil::TRP] [pDB3182] were grown for 4-days at 3O°C, 200rpm. Samples were spread onto YEPD-agar plates and grown to single colonies. Fifty randomly selected colonies were patched out in replica onto BMMD and YEPD plates and incubated at 30°C. Plasmid stability was scored as the percentage of colonies able to grow on both media.
Figure imgf000148_0001
Arrows indicate the direction of transcription relative to the LEU2 gene. DXYl containing pDB3175-pDB3182 were compared with DXYl Λrrpl pdil:. TRPl containing pDB3175-pDB3182 for the secretion of recombinant human albumin and transferrin (N413Q. N61 IQ). Figure 34 shows that following disruption of the genomic PDIl gene with TRPl to increase plasmid stability to 100% (Table 16), there was a reduced recombinant human albumin titre for PDIl inserted at the SnaBl-sϊtc. However, this is more than compensated for by the increased genetic stability following disruption of PDIl in the genome, which increases the reproducibility and reliability of heterologous protein secretion, resulting in a more useful industrial organism for application in consistent protein production, especially in prolonged cultivation, such as fill and draw fermentation or continuous culture fermentation campaigns. Furthermore, when PDIl was located at the A'crøl-site after REP 2 of pSAC35 (Example 12) there was no significant decrease in rHA titre following disruption of the genomic PDIl gene with TRPl to increase plasmid stability (Table 13). This suggests that the Xcm\~ site was preferred (but not essential) compared to the SVzαBI-site for PDIl on 2μm-based plasmids expressing rHA. However, PDIl expression can be modulated, for example by altering the length of the PDIl promoter (Example T) such that an increase in recombinant protein secretion is observed. In the above experiment the long PDIl promoter was used, which was not the preferred promoter length for optimal rHA secretion in the closely related high rHA producing strain, DS569, where a short promoter resulted in increased rHA secretion. In the case of genomic PDIl disruption in DXYl [pDB2977] containing a long PDIl promoter in PDIl at the A'cml-site after REP 2 of pSAC35 (Figure 19), the analysis included three individual isolates (not triplicates of a single isolate as shown in Figure 34), and demonstrated no decrease in rHA titre. Furthermore, the increased consistency and reproducibility is clearly demonstrated for these cultures following disruption of the genomic PDIl gene.
In Figure 35 a large increase in recombinant transferrin secretion was observed between DXYl [pDB2931] without PDIl on the pSAC35-based vector (which itself was not 100% stable), and strains containing plasmids pDB3179-pDB3182, with PDIl at the SnaBllNotl-siXt. In DXYl Atrpl pdil -TRPl containing plasmids pDB3179-pDB3182, PDIl on the plasmid was acting as the sole selectable marker and resulted in improved genetic stability (see Table 17) where 100% plasmid stability was observed without any decrease in recombinant transferrin secretion. Hence, by placing PDIl on the transferrin expression plasmid and disrupting PDIl in the genome, the overall effect has been to increase secretion of the recombinant protein and also to improve the genetic stability of the production organism

Claims

1. A method for producing a desired protein (such as a desired heterologous protein) comprising:
(a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different; and
(b) culturing the host cell in a culture medium to obtain expression of the first, second and third genes.
2. The method of Claim 1 further comprising the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium.
3. The method of Claim 2 further comprising the step of lyophilising the thus purified protein.
4. The method of Claim 2 or 3 further comprising the step of formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
5. A method according to any preceding claim wherein one or both of the first or second chaperone proteins has a sequence of a fungal chaperone (optionally a yeast chaperone) or a mammalian chaperone (optionally a human chaperone).
6. A method according to any one of the preceding claims wherein one or both of the first and second chaperones each individually comprise the sequence of a protein encoded by any one of AHAl, CCT2, CCTS, CCT4, CCT5, CCT6, CCT7, CCT8, CNSl, CPR3, CPR6, EPSl, EROl, EUGl, FMOl, HCHl, HSPlO, HSP12, HSPl 04, HSP26, HSP30, HSP42, HSP60, HSP 78, HSP82, JEMl, MDJl, MDJ2, MPDl, MP D2, PDIl, PFDl, ABCl, APJl, ATPIl, ATP 12, BTTl, CDC37, CPR7, HSC82, KAR2, LHSl,
MGEl, MRSIl, NOBl, ECMlO, SSAl, SSA2, SSA3, SSA4, SSCl, SSE2, SILl, SLSl, UBI4, ORMl, 0RM2, PERl, PTC2, PSEl, HACl or truncated intronless HACl, TIM9, P AM 18 or TCPl or a variant or fragment of any one of these.
7. A method according to any one of the preceding claims wherein the first chaperone is protein disulphide isomerase.
8. A method according to any one of the preceding claims wherein the second chaperone is Orm2p or a variant or fragment thereof.
9. A method according to any one of the preceding claims wherein at least one, preferably both, of the first or second chaperones is encoded by a chromosomally integrated recombinant gene.
10. A method according to any one of the preceding claims wherein at least one, preferably both, of the first or second chaperones is encoded by a gene on a plasmid.
11. A method according to any preceding claim wherein the third gene which encodes the desired protein (such as a desired heterologous protein) is integrated in the chromosome of the host cell, or is provided on a plasmid within the host cell.
12. A method according to Claim 10 or 11 wherein the plasmid is, or is not, a 2μm-family plasmid.
13. A method according to Claim 12 wherein the plasmid comprises a gene encoding a protein comprising the sequence of the first chaperone protein and/or a gene encoding a protein comprising the sequence of the second chaperone protein, and a gene encoding a desired heterologous protein.
14. A method according to Claim 12 or 13 wherein the plasmid is a disintegration vector.
15. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) comprises a leader sequence effective to cause secretion from the host cell, such as yeast.
16. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) is a eucaryotic protein, or a fragment or variant thereof, optionally a vertebrate or a fungal (such as a yeast) protein.
17. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) is a commercially useful protein, such as a therapeutically, diagnostically, industrially, domestically or nutritionally useful protein.
18. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) comprises a sequence selected from albumin, a monoclonal antibody, an etoposide. a serum protein (such as a blood clotting factor, e.g. Factor VII. Factor VIII, Factor IX, Factor X and Factor XIII). antistasin, a tick anticoagulant peptide, transferrin, lactoferrin, endostatin. angiostatin. collagens. immunoglobulins, or immunoglobulin-based molecules or fragment of either (e.g. a dAb. Fab' fragments, F(ab')2, scAb, scFv or scFv fragment), a Kunitz domain protein, interferons, interleukins, ILl O. ILI l. IL2, interferon α species and sub-species, interferon β species and sub-species, interferon γ species and sub-species, leptin, CNTF, CNTFAχi5XAxokine™), ILl -receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanoλάrin-N. 5-helix, T20 peptide, Tl 249 peptide, HIV gp41, HIV gpl20, urokinase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-like peptides, insulin-like growth factor, calcitonin, growth hormone, transforming growth factor β, tumour necrosis factor, G-
CSF, GM-CSF, M-CSF, FGF, coagulation factors in both pre and active forms, including but not limited to plasminogen, fibrinogen, thrombin, pre- thrombin, pro-thrombin, von Willebrand's factor, αi -antitrypsin, plasminogen activators, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, aprotinin, amyloid precursor protein, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, a metabolite, an antibiotic, or a variant or fragment of any of the above.
19. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) comprises the sequence of albumin or a variant or fragment thereof.
20. A method according to any preceding claim wherein the desired protein (such as a desired heterologous protein) comprises the sequence of a transferrin family member, optionally transferrin or lactoferrin. or a variant or fragment thereof.
21. A method according to any preceding claim wherein the desired protein is a desired heterologous protein that comprises a fusion protein, such as a fusion protein of albumin or a transferrin family member or a λ'ariant or fragment of either, fused directly or indirectly to the sequence of another protein.
22. A plasmid comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein and a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein.
23. A plasmid according to Claim 22 wherein at least one, and optionally both, of the first and second chaperones are chaperones as defined by any one of Claims 5 to 8.
24. A plasmid according to Claim 22 or 23 further comprising a third recombinant gene, which third recombinant gene encodes a desired heterologous protein, optionally a desired heterologous protein as described by any one of Claims 15-21.
25. A plasmid according to any one of Claims 22 to 24 wherein the plasmid is a plasmid as defined by any one of Claims 12 to 14.
26. A host cell comprising a first recombinant gene encoding a protein comprising the sequence of a first chaperone protein, a second recombinant gene encoding a protein comprising the sequence of a second chaperone protein and a third gene, such as a third recombinant gene, encoding a desired protein (such as a desired heterologous protein), wherein the first and second chaperones are different.
27. A host cell according to Claim 26. which host cell is a host cell as defined by any one of Claims 5 to 21.
28. A host cell comprising a plasmid as defined by any one of Claims 22-25.
29. A method according to any one of Claims 1 to 21 or a host cell according to any one of Claims 26 to 28 wherein the host cell is a bacterial or yeast host cell.
30. A method or a host cell according to Claim 29 wherein the host cell is a yeast cell, optionally a member of the Saccharomyces, Kluyveromyces, Arxula, Yarrowia, Candida, Schizosaccharomyces, Debaryomyces,
Xanthophyllomyces, Geotrichum, Ashbya, Hortaea, Schwanniomyces, Trichosporon, Xanthophyllomyces, or Pichia genus, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris, Pichia membranaefaciens, Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha (also known as Pichia augusta), Arxula adeninivorans, Yarrowia lipolyϊica, Candida boidinii Candida utilis or Schizosaccharomyces pombe.
31. A host cell according to Claim 27, or as defined by Claim 10 or 11, in which the plasmid is a 2μm-family plasmid and wherein:
(a) the plasmid is based on pSRl, pSB3 or pSB4 and the host cell is Zygosaccharomyces rouxii;
(b) the plasmid is based on pSBl or pSB2 and the host cell is Zygosaccharomyces bailii; (c) the plasmid is based on pSMl and the host cell is Zygosaccharomyces ferm entati;
(d) the plasmid is based on pKDl and the host cell is Kluyveromyces drosophilarum; (e) the plasmid is based on pPMl and the host cell is Pichia membranaefaciens; or
(f) the plasmid is based on the 2μm plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.
32. A host cell according to Claim 31 in which the plasmid is based on the 2μm plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.
33. A method for producing desired protein (such as a desired heterologous protein) comprising:
(a) providing a host cell comprising a first recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the first and second genes are not present within the host cell on the same 2μm-family plasmid; and
(b) culturing the host cell in a culture medium to obtain expression of the first and second genes.
34. A method according to Claim 33 wherein either or both of the first and second genes are expressed from a plasmid, and optionally from the same plasmid.
35. A method according to Claim 33 or 34 wherein the first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof is located on a plasmid.
36. A method according to Claim 33 wherein either or both of the first and second genes are integrated into the chromosome of the host cell.
37. A method according to Claim 33 or 36 wherein the first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof is integrated into the chromosome of the host cell.
38. A method according to any one of Claims 33 to 37 further comprising the step of purifying the thus expressed desired protein (such as a desired heterologous protein) from the cultured host cell or the culture medium.
39. A method according to Claim 38 further comprising the step of formulating the purified desired protein (such as a desired heterologous protein) with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
40. A host cell comprising a first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the first and second recombinant genes are not present within the host cell on the same 2 μm -family plasmid.
41. Use of a recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof to increase the expression of a desired protein (such as a desired heterologous protein) in a host cell, with the proviso that the recombinant gene encoding a protein comprising the sequence of Orm2p or a variant or fragment thereof and the gene encoding the desired protein (such as a desired heterologous protein) are not co- expressed within the host cell from the same 2μm-family plasmid.
42. A plasmid comprising a first recombinant gene encoding a protein comprising the sequence of 0rm2p or a variant or fragment thereof and a second gene, such as a second recombinant gene, encoding a desired protein (such as a desired heterologous protein), with the proviso that the plasmid is not a 2μm-family plasmid.
43. A method, use, host cell or plasmid according to any one of Claims 33 to 42 wherein the desired protein (such as a desired heterologous protein) is as defined in any one of Claims 15 to 21.
44. A host cell comprising a plasmid, the plasmid comprising a gene that encodes an essential chaperone wherein, in the absence of the plasmid, the host cell is unable to produce the chaperone, the plasmid further comprising a recombinant gene encoding a desired heterologous protein, such as a desired heterologous protein as defined in any one of Claims 15 to 21.
45. A host cell according to Claim 44 wherein the essential chaperone is a eucaryotic chaperone.
46. A host cell according to Claim 44 wherein the essential chaperone is a yeast chaperone.
47. A host cell according to Claim 44 wherein the essential chaperone is a yeast chaperone comprises the sequence of a protein encoded by a gene selected from CCT2, CCT3, CCT4, CCT5.. CCT6, CCT7, CCT8.. CNSl, EROl, HSPlO, HSP 60. PDIl, CDC37, KAR2, MGEl, MRSlL NOBl, SSCl, PSEl, TIM9, P AM 18 and TCPl or a variant or fragment of any one of these..
48. A host cell according to Claim 44 wherein the essential chaperone is protein disulphide isomerase.
49. A host cell according to any one of Claims 44 to 48 wherein, in the absence of the plasmid, the host cell is inviable.
50. A host cell according to Claim 49 wherein, in the absence of the plasmid, the host cell is inviable and cannot be made viable by growing the host cell in a growth medium and making nutrient additions or modifications to that growth medium, and preferably cannot be made viable by growing the host cell in a growth medium and making any additions or modifications to that growth medium.
51. A plasmid comprising, as the sole yeast selectable marker, a gene encoding a chaperone that is essential to the viability of a yeast host cell, in the sense that when the gene or genes encoding the essential chaperone are deleted or inactivated in a yeast host cell, then the host cell is inviable in culture and the deficiency cannot be complemented by additions or modifications to the culture medium.
52. A plasmid according to Claim 51 wherein the gene encoding the essential chaperone is the sole selectable marker encoded by the plasmid.
53. A plasmid according to Claim 52 wherein the essential chaperone is a eucaryotic chaperone.
54. A plasmid according to Claim 52 wherein the essential chaperone is a yeast chaperone.
55. A plasmid according to Claim 52 wherein the essential chaperone is a yeast chaperone comprises the sequence of a protein encoded by a gene selected from CCT2, CCTS, CCT4, CCT5, CCT6, CCTl, CCTB, CNSl, EROl, HSPlO. HSP60, PDIl, CDCSl, KΛR2, MGEl, MRSIl, NOBl, SSCl, PSEl, TIM9, PAMl 8 and TCPl or a variant or fragment of any one of these.
56. A plasmid according to Claim 52, wherein the essential chaperone is protein disulphide isomerase or Pselp.
57. A plasmid according to any one of Claims 51 to 56 further comprising a gene encoding a desired heterologous protein, such as a desired heterologous protein defined by any one of Claims 15 to 21.
58. A plasmid according to any one of Claims 51 to 57 which is a 2μm-family plasmid.
59. A plasmid according to any one of Claims 51 to 58 which is a plasmid as defined by any one of Claims 22 to 25.
60. A host cell according to any one of Claims 44 to 50 in which the plasmid is a plasmid according to any one of Claims 51 to 59.
61. A host cell according to Claim 28 wherein a chaperone encoded by the plasmid is an essential gene.
62. A host cell according to Claim 60 wherein, in the absence of the plasmid. the host cell does not produce the chaperone.
63. A method according to Claim 1 wherein the host cell is a host cell as defined by Claim 61 or 62.
64. A method according to Claim 63 wherein the step (b) of Claim 1 involves culturing the host cell in non-selective media, such as rich or complex media.
65. A method for producing a desired recombinant protein comprising the steps of: providing a host cell comprising a plasmid, the plasmid comprising a first recombinant gene that encodes a chaperone that is essential to the viability of the host cell (the "essential chaperone") and wherein, in the absence of the plasmid. the host cell is unable to produce the essential chaperone, which host cell is optionally a host cell as defined by any one of Claims 44 to 50 or 60 to 62, and further in which the host cell comprises a gene that encodes a desired protein; culturing the host cell in a culture medium under conditions that allow the expression of the essential chaperone and the desired protein; and optionally isolating the thus expressed desired protein from the cultured host cell or the culture medium; and optionally purifying the thus isolated desired protein to a commercially acceptable level of purity; and further optionally, lyophilising the thus purified protein or formulating the purified desired protein with a carrier or diluent (such as a pharmaceutically acceptable carrier or diluent); and optionally presenting the thus formulated desired protein in a unit dosage form.
66. The method of Claim 65 wherein the step of culturing the host cell involves culturing the host cell in a non-selective media, such as a complex or rich media.
67. A method for producing a desired protein (such as a desired heterologous protein) comprising the steps of:
(a) providing a host cell as defined by any one of Claims 44 to 50; and
(b) culturing the host cell in a culture medium under conditions that allow the expression of the essential chaperone and the desired protein.
68. The method of Claim 67 wherein the host cell comprises a plasmid as defined by any one of Claims 51 to 59.
69. The method of Claim 67 or 68 wherein step (b) of the method of Claim 67 is performed by culturing the host cell in a non-selective medium, such as a rich or complex medium.
70. Use of a polynucleotide comprising a sequence that encodes a chaperone that is essential to the viability of a host cell (the "essential chaperone"), to increase the stability of a plasmid in the host cell by integration of the polynucleotide into the plasmid to produce a modified plasmid, wherein the host cell is unable to produce the essential chaperone in the absence of the modified plasmid.
71. Use according to Claim 70 to increase the stability of a plasmid in the host cell when the host cell is grown under non-selective conditions, such as in a rich or complex medium.
72. Use according to Claim 70 or 71 wherein the plasmid is a 2μm-family plasmid.
73. Use according to any one of Claims 70 to 72 wherein the plasmid further comprises a gene encoding a desired heterologous protein, such as a desired heterologous protein as defined in any one of Claims 15 to 21.
74. Use according to any one of Claims 70 to 73 wherein the essential chaperone is a eucaryotic chaperone.
75. Use according to any one of Claims 70 to 73 wherein the essential chaperone is a yeast chaperone.
76. Use according to any one of Claims 70 to 73 wherein the essential chaperone is a yeast chaperone comprises the sequence of a protein encoded by a gene selected from CCT2, CCT3, CCT4, CCT5, CCT6, CCTl, CCT8, CNSJ, EROl, HSPlO, HSP 60, PDIl, CDC37, KAR2,
MGEl, MRSIl, NOBl, SSCl, PSEl, TIM9, PAMl 8 and TCPl or a variant or fragment of any one of these.
77. Use according to any one of Claims 70 to 73 wherein the essential protein is a chaperone, such as protein disulphide isomerase or Pselp.
78. Use according to any one of Claims 70 to 77 to simultaneously increase the stability of a plasmid in the host cell and increase the ability of the host cell to produce protein product.
79. Use according to Claim 78 wherein the protein product is an endogenously encoded protein or a heterologous protein, such as a heterologous protein as defined in any one of Claims 15 to 21.
80. Use according to Claim 79 wherein the protein product is a heterologous protein that is encoded by a recombinant gene that has been integrated into the chromosome of the host cell.
81. Use according to Claim 79 wherein the protein product is a heterologous protein that is encoded by a recombinant gene that is present on a plasmid in the host cell.
82. Use according to Claim 81 wherein the plasmid that comprises the recombinant gene that encodes the heterologous protein is the same plasmid as the modified plasmid that comprises the polynucleotide that encodes the essential chaperone.
83. A host cell, plasmid, method or use according to any one of Claims 44 to 82 wherein the host cell is a bacterial or yeast host cell.
84 A host cell, plasmid, method or use according to Claim 83 wherein the host cell is a yeast cell, optionally a member of the Saccharomyces,
Figure imgf000165_0001
Arxula, Yarrowia, Candida, Schizosaccharomyces, Debaryomyces, Xanthophyllomyces, Geotrichum, Ashbya, Hortaea,
Schwanniomyces, Trichosporon, Xanthophyllomyces, or Pichia genus, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoήs, Pichia membranaefaciens, Zygosaccharomyces rouxii,
Zygosaccharomyces bailii, Zygosaccharomyces fermentati ', Kluyveromyces drosphilarum, Pichia methanolica, Hansenula polymorpha (also known as
Pichia auguslά), Arxula adeninivorans, Yarrowia lipolytica, Candida boidinii Candida utilis or Schizosaccharomyces pombe.
85. A host cell, plasmid, method or use according to Claim 84, in which the plasmid is a 2μm-family plasmid and wherein: (a) the plasmid is based on pSRl. pSB3 or pSB4 and the host cell is Zygosaccharomyces rouxii,
(b) the plasmid is based on pSBl or pSB2 and the host cell is Zygosaccharomyces bailli, (c) the plasmid is based on pSMl and the host cell is
Zygosaccharomyces fermentati.
(d) the plasmid is based on pKDl and the host cell is Kluyveromyces drosophilarum;
(e) the plasmid is based on pPMl and the host cell is Pichia membranaefaciens, or
(f) the plasmid is based on the 2μm plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.
86. A host cell, plasmid, method or use according to Claim 85 in which the plasmid is based on the 2μm plasmid and the host cell is Saccharomyces cerevisiae or Saccharomyces carlsbergensis.
87. Use of a polynucleotide comprising the sequence of a promoter operably connected to a coding sequence encoding a chaperone for increasing the expression of a desired protein (such as a desired heterologous protein) in a host cell by expression of the polynucleotide sequence within the host cell, wherein the promoter is characterised in that it achieves a lower level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter, optionally wherein the use is a use as defined in any one of
Claims 70 to 82.
88. A method for producing a desired protein (such as a desired heterologous protein) comprising the steps of: (a) providing a host cell comprising a recombinant gene that comprising the sequence of promoter operably connected to a coding sequence encoding a chaperone, the promoter being characterised in that it achieves a lower level of expression of the chaperone than would be achieved if the coding sequence were to be operably connected to its naturally occurring promoter, and the host cell further comprising a gene, such as a recombinant gene, encoding a desired protein (such as a desired heterologous protein); and
(b) culturing the host cell in a under conditions that allow the expression of the chaperone and the desired protein (such as a desired heterologous protein);
optionally wherein the method is a method according to any preceding method claim.
89. The method of Claim 88 further comprising the step of purifying the desired protein (such as a desired heterologous protein) produced in step (b); and optionally lyophilising the thus purified protein.
90. The method of Claim 89 further comprising the step of formulating the purified or lyophilised desired protein (such as a desired heterologous protein) with a carrier or diluent.
91. The method of Claim 90 further comprising the step of presenting the thus formulated protein in a unit dosage form.
PCT/GB2005/005085 2003-12-23 2005-12-23 Gene expression technique WO2006067511A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
CN200580048615.0A CN101128587B (en) 2003-12-23 2005-12-23 Gene expression technique
EP05823505.2A EP1831375B1 (en) 2004-12-23 2005-12-23 Gene expression technique
JP2007547668A JP5631533B2 (en) 2004-12-23 2005-12-23 Gene expression technology
AU2005317828A AU2005317828A1 (en) 2004-12-23 2005-12-23 Gene expression technique
US11/722,539 US9057061B2 (en) 2003-12-23 2005-12-23 Gene expression technique
US13/474,313 US8969064B2 (en) 2003-12-23 2012-05-17 Gene expression technique
US14/605,314 US20160186192A1 (en) 2003-12-23 2015-01-26 Gene expression technique

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBPCT/GB2004/005435 2004-12-23
PCT/GB2004/005435 WO2005061719A1 (en) 2003-12-23 2004-12-23 2-micron family plasmid and use thereof
PCT/GB2004/005462 WO2005061718A1 (en) 2003-12-23 2004-12-23 Gene expression technique
GBPCT/GB2004/005462 2004-12-23

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
PCT/GB2004/005435 Continuation-In-Part WO2005061719A1 (en) 2003-12-23 2004-12-23 2-micron family plasmid and use thereof
PCT/GB2004/005462 Continuation-In-Part WO2005061718A1 (en) 2003-12-23 2004-12-23 Gene expression technique

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US11/722,539 A-371-Of-International US9057061B2 (en) 2003-12-23 2005-12-23 Gene expression technique
US13/474,313 Division US8969064B2 (en) 2003-12-23 2012-05-17 Gene expression technique

Publications (1)

Publication Number Publication Date
WO2006067511A1 true WO2006067511A1 (en) 2006-06-29

Family

ID=35848381

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2005/005085 WO2006067511A1 (en) 2003-12-23 2005-12-23 Gene expression technique

Country Status (6)

Country Link
US (3) US9057061B2 (en)
EP (2) EP1831375B1 (en)
JP (1) JP5631533B2 (en)
AU (1) AU2005317828A1 (en)
DK (1) DK2330200T3 (en)
WO (1) WO2006067511A1 (en)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006136831A2 (en) * 2005-06-22 2006-12-28 Novozymes Delta Limited Gene expression technique
WO2009019314A1 (en) 2007-08-08 2009-02-12 Novozymes A/S Transferrin variants and conjugates
WO2010092335A1 (en) * 2009-02-10 2010-08-19 Ucb Pharma S.A. Method for producing protein
WO2010092135A2 (en) 2009-02-11 2010-08-19 Novozymes Biopharma Uk Ltd. Albumin variants and conjugates
WO2010133668A2 (en) 2009-05-20 2010-11-25 Fh Campus Wien Eukaryotic host cell comprising an expression enhancer
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
WO2015158808A2 (en) 2014-04-17 2015-10-22 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell engineered to overexpress helper proteins
WO2015158800A1 (en) 2014-04-17 2015-10-22 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell for expressing proteins of interest
US9315565B2 (en) 2009-02-10 2016-04-19 Ucb Pharma, S.A. Method for producing protein
CN105985428A (en) * 2015-02-10 2016-10-05 许健 Procoagulant function of human recombinant blood coagulation factor XIII
WO2017112847A1 (en) 2015-12-22 2017-06-29 Albumedix A/S Improved protein expression strains
US9944691B2 (en) 2012-03-16 2018-04-17 Albumedix A/S Albumin variants
EP2304045B1 (en) * 2008-07-28 2018-05-09 evocatal GmbH Chaperone toolbox
WO2018234349A1 (en) 2017-06-20 2018-12-27 Albumedix Ltd Improved protein expression strains
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
WO2019185535A1 (en) * 2018-03-26 2019-10-03 Novozymes A/S Fungal chaperone proteins
US10501524B2 (en) 2012-11-08 2019-12-10 Albumedix Ltd Albumin variants
US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
US10696732B2 (en) 2009-10-30 2020-06-30 Albumedix, Ltd Albumin variants
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
EP3716274A1 (en) 2019-03-29 2020-09-30 Ajinomoto Co., Inc. Control device, control method, computer program, and method for producing organic compound
EP4194560A1 (en) * 2021-12-08 2023-06-14 European Molecular Biology Laboratory Improved production of secreted proteins in fungal cells
CN118460394A (en) * 2024-07-12 2024-08-09 通化安睿特生物制药股份有限公司 Engineering bacterium for preparing transferrin and application thereof

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ534492A (en) * 2002-02-07 2009-10-30 Novozymes Delta Ltd Albumin-fused kunitz domain peptides
US20080044853A1 (en) * 2004-06-21 2008-02-21 Novozymes A/S Stably Maintained Multiple Copies of at Least Two Orf in the Same Orientation
US9447422B2 (en) 2012-12-06 2016-09-20 Synthetic Genomics, Inc. Autonomous replication sequences and episomal DNA molecules
US20150337274A1 (en) * 2012-12-17 2015-11-26 Ming-Tang Chen Pmt2-, och1-, pmt5- mutant cells
CN104937599B (en) 2013-02-28 2018-01-23 株式会社日立高新技术 Data analysis device and its method
CN107988289A (en) * 2017-11-20 2018-05-04 广东海纳川生物科技股份有限公司 A kind of method for improving Pichia pastoris secreting, expressing plectasin expression quantity

Citations (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4302386A (en) 1978-08-25 1981-11-24 The Ohio State University Antigenic modification of polypeptides
EP0060057A1 (en) 1981-02-25 1982-09-15 Genentech, Inc. Expression of polypeptides in yeast
EP0073646A2 (en) 1981-08-28 1983-03-09 Genentech, Inc. Construction of DNA sequences and their use for microbial production of proteins, in particular human serum albumin
EP0074661A2 (en) 1981-09-14 1983-03-23 Union Carbide Corporation Process for producing a polymer water-in-oil emulsion
EP0251744A2 (en) 1986-06-27 1988-01-07 Delta Biotechnology Limited Process for the genetic modification of yeast
EP0258067A2 (en) 1986-08-29 1988-03-02 Delta Biotechnology Limited Yeast promoter
EP0286424A1 (en) 1987-04-09 1988-10-12 Delta Biotechnology Limited Yeast vector
EP0293793A2 (en) 1987-06-01 1988-12-07 Takeda Chemical Industries, Ltd. Polypeptide and production thereof
EP0319067A1 (en) 1987-11-18 1989-06-07 Gist-Brocades N.V. Process for the purification of serum albumin
WO1990001063A1 (en) 1988-07-23 1990-02-08 Delta Biotechnology Limited New secretory leader sequences
EP0366400A2 (en) 1988-10-26 1990-05-02 Tonen Corporation DNA encoding human serum albumin a (HSA), plasmids and hosts containing such DNA, and the production of HSA
WO1990013653A1 (en) 1989-04-29 1990-11-15 Delta Biotechnology Limited Fusion proteins containing n-terminal fragments of human serum albumin
EP0431880A1 (en) 1989-12-05 1991-06-12 Delta Biotechnology Limited Mutant fungal strain detection
EP0464590A2 (en) 1990-06-25 1992-01-08 The Green Cross Corporation Inhibition of coloration of human serum albumin
WO1992004367A1 (en) 1990-09-12 1992-03-19 Delta Biotechnology Limited Separation of proteins and dyes
EP0509841A2 (en) 1991-04-18 1992-10-21 Tonen Corporation Co-expression system of protein disulfide isomerase gene and useful polypeptide gene and process for producing the polypeptide using its system
WO1993025676A1 (en) 1992-06-12 1993-12-23 Merck & Co., Inc. METHOD FOR INCREASING PRODUCTION OF DISULFIDE BONDED RECOMBINANT PROTEINS BY $i(SACCHAROMYCES CEREVISIAE)
WO1994004687A1 (en) 1992-08-14 1994-03-03 Rhone-Poulenc Rorer S.A. Modified fungal cells and method for producing recombinant products
WO1995023857A1 (en) 1994-03-05 1995-09-08 Delta Biotechnology Limited Yeast strains and modified albumins
WO1995033833A1 (en) 1994-06-07 1995-12-14 Delta Biotechnology Limited Yeast strains
WO1996037515A1 (en) 1995-05-25 1996-11-28 Delta Biotechnology Limited Process of high purity albumin production
WO2000044772A2 (en) 1999-01-30 2000-08-03 Delta Biotechnology Limited Human serum albumin
WO2001079442A2 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc. Albumin fusion proteins
US6451559B1 (en) 1997-09-05 2002-09-17 Pentapharm Ag Expression vector for improved production of polypeptides in yeast
WO2003057897A2 (en) * 2002-01-07 2003-07-17 European Molecular Biology Laboratory Recombinant protein expression
WO2003066824A2 (en) 2002-02-07 2003-08-14 Aventis Behring Gmbh Albumin-fused kunitz domain peptides
US20030221201A1 (en) 2001-08-30 2003-11-27 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
US20030226155A1 (en) 2001-08-30 2003-12-04 Biorexis Pharmaceutical Corporation Modified transferrin-antibody fusion proteins
WO2004009819A2 (en) 2002-07-23 2004-01-29 Delta Biotechnology Limited Polypeptides with a signal sequence comprising an fivsi motif and olynucleotides encoding therefor
WO2004083245A2 (en) 2003-03-15 2004-09-30 Delta Biotechnology Limited Agent comprise an albumin-like first polypeptide bound to a second polypeptide
WO2005061719A1 (en) 2003-12-23 2005-07-07 Delta Biotechnology Limited 2-micron family plasmid and use thereof
WO2005061718A1 (en) 2003-12-23 2005-07-07 Delta Biotechnology Limited Gene expression technique
WO2005078105A2 (en) * 2004-02-06 2005-08-25 Medical Research Council Selection markers useful for heterologous protein expression

Family Cites Families (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU600885B2 (en) * 1984-05-25 1990-08-30 Zymogenetics Inc. Stable DNA constructs
US4931373A (en) 1984-05-25 1990-06-05 Zymogenetics, Inc. Stable DNA constructs for expression of α-1 antitrypsin
WO1989007140A1 (en) 1988-02-05 1989-08-10 Commonwealth Scientific And Industrial Research Or Gene expression system (particularly for rotavirus vp7 protein) involving a foreign signal peptide and optionally a transmembrane anchor sequence
JPH0760629B2 (en) 1989-05-09 1995-06-28 和泉電気株式会社 circuit protector
JPH03114047A (en) 1989-09-28 1991-05-15 Sharp Corp Rotation control method for heat roller
MY106504A (en) * 1990-07-13 1995-06-30 Transkaryotic Therapies Inc Library screening method.
JPH07502723A (en) 1991-02-08 1995-03-23 ザ・ユニバーシテイ・オブ・バーモント・アンド・ステイト・アグリカルチユラル・カレツジ Recombinant transferrin, transferrin half-molecules, and mutants thereof
ZA932568B (en) 1992-04-24 1993-11-12 Baylor College Midecine A Non Production of recombinant human lactoferrin
WO1994003617A1 (en) 1992-07-29 1994-02-17 Unilever N.V. Process for producing anti-freeze peptides
DE69329202T2 (en) 1992-10-02 2001-03-29 Research Corp. Technologies, Inc. METHODS OF INCREASING THE SECRETION OF OVEREXPRESSED PROTEINS
ATE346941T1 (en) 1996-09-04 2006-12-15 Suntory Ltd PROTEIN DISULFIDE ISOMERASE GENE OF A METHYLOTROPHIC YEAST STRAIN
GB9703406D0 (en) 1997-02-19 1997-04-09 Chiron Spa Expression of heterologous proteins
AU8040798A (en) 1997-06-13 1998-12-30 Stichting Instituut Voor Dierhouderij En Diergezondheid Increased production of proteins by using chaperone-like proteins
US6210683B1 (en) * 1997-09-05 2001-04-03 Merck & Co., Inc. Stabilizers containing recombinant human serum albumin for live virus vaccines
EP1077263A1 (en) 1999-07-29 2001-02-21 F.Hoffmann-La Roche Ag Process for producing natural folded and secreted proteins by co-secretion of chaperones
JP4396078B2 (en) * 1999-12-17 2010-01-13 チッソ株式会社 Microorganism incubator and microorganism medium using the same
EP1266018B1 (en) 2000-03-24 2008-05-07 Genencor International, Inc. Production of secreted proteins by recombinant eukaryotic cells
US6358733B1 (en) 2000-05-19 2002-03-19 Apolife, Inc. Expression of heterologous multi-domain proteins in yeast
FI120310B (en) 2001-02-13 2009-09-15 Valtion Teknillinen An improved method for producing secreted proteins in fungi
DE10121235A1 (en) 2001-04-30 2002-10-31 Roche Diagnostics Gmbh Process for the expression of proteins in in vitro translation systems with co-expression of folding helper proteins
CA2449747C (en) 2001-06-22 2010-04-13 F. Hoffmann-La Roche Ag Use of fkbp chaperones as expression tool
WO2003020480A1 (en) * 2001-08-21 2003-03-13 Alexander Ivanovich Kryukov Device for machining articles in the form of rotational bodies
DE10145694A1 (en) 2001-09-17 2003-04-03 Roche Diagnostics Gmbh Process for increasing the solubility, expression rate and activity of proteins during recombinant production
JP2005534330A (en) 2002-08-07 2005-11-17 デルタ バイオテクノロジー リミテッド Albumin fusion ciliary neurotrophic factor
DE10252245A1 (en) 2002-11-07 2004-05-27 Prof. Dr. Danilo Porro Università degli Studi di Milano-Bicocca Dipartimento die Biotechnologie e Bioscienze Producing a protein useful as pharmaceuticals, e.g. medicine and vaccine, or in food or paper production, comprises expressing and secreting a protein expressed by Zygosaccharomyces bailii strain
US6939676B2 (en) * 2002-12-30 2005-09-06 The United States Of America As Represented By The Secretary Of Argriculture Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics
US7244616B2 (en) 2003-06-27 2007-07-17 Bayer Pharmaceuticals Corporation Use of molecular chaperones for the enhanced production of secreted, recombinant proteins in mammalian cells
US7226781B1 (en) 2003-07-24 2007-06-05 Belyaev Alexander S Chaperone expression genomes
JPWO2005037316A1 (en) * 2003-10-20 2006-12-28 株式会社ロコモジェン Method for inhibiting secretase activity
JP4988337B2 (en) 2004-05-21 2012-08-01 タカラバイオ株式会社 Method for producing polypeptide
JP2008507294A (en) 2004-07-26 2008-03-13 ダウ グローバル テクノロジーズ インコーポレイティド Method for improved protein expression by strain genetic manipulation

Patent Citations (41)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4302386A (en) 1978-08-25 1981-11-24 The Ohio State University Antigenic modification of polypeptides
EP0060057A1 (en) 1981-02-25 1982-09-15 Genentech, Inc. Expression of polypeptides in yeast
EP0073646A2 (en) 1981-08-28 1983-03-09 Genentech, Inc. Construction of DNA sequences and their use for microbial production of proteins, in particular human serum albumin
EP0074661A2 (en) 1981-09-14 1983-03-23 Union Carbide Corporation Process for producing a polymer water-in-oil emulsion
EP0251744A2 (en) 1986-06-27 1988-01-07 Delta Biotechnology Limited Process for the genetic modification of yeast
EP0258067A2 (en) 1986-08-29 1988-03-02 Delta Biotechnology Limited Yeast promoter
EP0286424A1 (en) 1987-04-09 1988-10-12 Delta Biotechnology Limited Yeast vector
EP0293793A2 (en) 1987-06-01 1988-12-07 Takeda Chemical Industries, Ltd. Polypeptide and production thereof
EP0319067A1 (en) 1987-11-18 1989-06-07 Gist-Brocades N.V. Process for the purification of serum albumin
WO1990001063A1 (en) 1988-07-23 1990-02-08 Delta Biotechnology Limited New secretory leader sequences
EP0366400A2 (en) 1988-10-26 1990-05-02 Tonen Corporation DNA encoding human serum albumin a (HSA), plasmids and hosts containing such DNA, and the production of HSA
WO1990013653A1 (en) 1989-04-29 1990-11-15 Delta Biotechnology Limited Fusion proteins containing n-terminal fragments of human serum albumin
EP0431880A1 (en) 1989-12-05 1991-06-12 Delta Biotechnology Limited Mutant fungal strain detection
EP0464590A2 (en) 1990-06-25 1992-01-08 The Green Cross Corporation Inhibition of coloration of human serum albumin
WO1992004367A1 (en) 1990-09-12 1992-03-19 Delta Biotechnology Limited Separation of proteins and dyes
EP0509841A2 (en) 1991-04-18 1992-10-21 Tonen Corporation Co-expression system of protein disulfide isomerase gene and useful polypeptide gene and process for producing the polypeptide using its system
WO1993025676A1 (en) 1992-06-12 1993-12-23 Merck & Co., Inc. METHOD FOR INCREASING PRODUCTION OF DISULFIDE BONDED RECOMBINANT PROTEINS BY $i(SACCHAROMYCES CEREVISIAE)
US6291205B1 (en) 1992-06-12 2001-09-18 Merck & Co., Inc. Method of increasing production of disulfide bonded recombinant proteins by saccharomyces cerevisiae
EP0746611A1 (en) 1992-06-12 1996-12-11 Merck & Co. Inc. METHOD FOR INCREASING PRODUCTION OF DISULFIDE BONDED RECOMBINANT PROTEINS BY $i(SACCHAROMYCES CEREVISIAE)
WO1994004687A1 (en) 1992-08-14 1994-03-03 Rhone-Poulenc Rorer S.A. Modified fungal cells and method for producing recombinant products
US5728553A (en) 1992-09-23 1998-03-17 Delta Biotechnology Limited High purity albumin and method of producing
WO1995023857A1 (en) 1994-03-05 1995-09-08 Delta Biotechnology Limited Yeast strains and modified albumins
WO1995033833A1 (en) 1994-06-07 1995-12-14 Delta Biotechnology Limited Yeast strains
WO1996037515A1 (en) 1995-05-25 1996-11-28 Delta Biotechnology Limited Process of high purity albumin production
US6451559B1 (en) 1997-09-05 2002-09-17 Pentapharm Ag Expression vector for improved production of polypeptides in yeast
WO2000044772A2 (en) 1999-01-30 2000-08-03 Delta Biotechnology Limited Human serum albumin
WO2001079442A2 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc. Albumin fusion proteins
WO2001079271A1 (en) 2000-04-12 2001-10-25 Principia Pharmaceutical Corporation Albumin fusion proteins
WO2001079480A1 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc. Albumin fusion proteins
WO2001079443A2 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc. Albumin fusion proteins
WO2001079258A1 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc. Albumin fusion proteins
WO2001079444A2 (en) 2000-04-12 2001-10-25 Human Genome Sciences, Inc Albumin fusion proteins
US20030221201A1 (en) 2001-08-30 2003-11-27 Biorexis Pharmaceutical Corporation Modified transferrin fusion proteins
US20030226155A1 (en) 2001-08-30 2003-12-04 Biorexis Pharmaceutical Corporation Modified transferrin-antibody fusion proteins
WO2003057897A2 (en) * 2002-01-07 2003-07-17 European Molecular Biology Laboratory Recombinant protein expression
WO2003066824A2 (en) 2002-02-07 2003-08-14 Aventis Behring Gmbh Albumin-fused kunitz domain peptides
WO2004009819A2 (en) 2002-07-23 2004-01-29 Delta Biotechnology Limited Polypeptides with a signal sequence comprising an fivsi motif and olynucleotides encoding therefor
WO2004083245A2 (en) 2003-03-15 2004-09-30 Delta Biotechnology Limited Agent comprise an albumin-like first polypeptide bound to a second polypeptide
WO2005061719A1 (en) 2003-12-23 2005-07-07 Delta Biotechnology Limited 2-micron family plasmid and use thereof
WO2005061718A1 (en) 2003-12-23 2005-07-07 Delta Biotechnology Limited Gene expression technique
WO2005078105A2 (en) * 2004-02-06 2005-08-25 Medical Research Council Selection markers useful for heterologous protein expression

Non-Patent Citations (126)

* Cited by examiner, † Cited by third party
Title
"Genetics and Medical Applications", 1996, ACADEMIC PRESS, INC., article "All About Albumin: Biochemistry", pages: 170 - 181
ALI ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 24066
ALI ET AL., JBIOL CHEM, vol. 274, 1999, pages 24066
ANONYMOUS: "ProFold vector Technology", INTERNET ARTICLE, 16 August 2004 (2004-08-16), XP002370508, Retrieved from the Internet <URL:http://web.archive.org/web/20040816225908/http://www.abvector.com/technology.htm> [retrieved on 20060302] *
BACHMAIR; RUIS, MONATSHEFTE FÜR CHEMIE, vol. 115, 1984, pages 1229
BAO ET AL., YEAST, vol. 16, 2000, pages 329 - 341
BAO; FUKUHARA, GENE, vol. 272, 2001, pages 103 - 110
BECKER; CRAIG, EUR. J. BIOCHEM., vol. 219, 1994, pages 11 - 23
BECKER; GUARENTE, METHODS ENZYMOL., vol. 194, 1990, pages 182
BEGGS, NATURE, vol. 275, 1978, pages 104 - 109
BERENT ET AL., BIOTECH., vol. 3, 1985, pages 208
BOTSTEIN ET AL., GENE, vol. 8, 1979, pages 17 - 24
BROACH ET AL., COLD SPRING HARBOR SYMP. QUANT. BIOL., vol. 47, 1982, pages 1165 - 1174
BROACH; HICKS, CELL, vol. 21, 1980, pages 501
BULLEID; FREEDMAN, NATURE, vol. 335, 1988, pages 649 - 51
CAMERON ET AL., NUCL. ACIDS RES., vol. 4, 1977, pages 1429
CASHMORE ET AL., MOL. GEN. GENET., vol. 203, 1986, pages 154 - 162
CHEN ET AL., CELL, vol. 69, 1992, pages 647
CHINERY; HINCHLIFFE, CURR. GENET., vol. 16, 1989, pages 21
CHINERY; HINCHLIFFE, CURR. GENET., vol. 16, 1989, pages 21 - 25
CHOW ET AL., J. CELL. SCI., vol. 101, no. 3, 1992, pages 709 - 719
COHEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 69, 1972, pages 2110
CREIGHTON ET AL., J. MOL. BIOL., vol. 142, 1980, pages 43 - 62
DEMOLDER ET AL., J. BIOTECHNOL., vol. 32, 1994, pages 179 - 189
DOCKAL, M. ET AL., J. BIOL. CHEM., vol. 274, 1999, pages 29303 - 29310
EDMAN ET AL., NATURE, vol. 317, 1985, pages 267 - 270
ELBLE, BIOTECHNIQUES, vol. 13, 1992, pages 18
FARQUHAR ET AL., GENE, vol. 108, 1991, pages 81 - 89
FINNIS ET AL., EUR. J. BIOCHEM., vol. 212, 1993, pages 201 - 210
FRAND; KAISER, MOLECULAR CELL, vol. 1, 1998, pages 161 - 170
FREEDMAN ET AL., BIOCHEM. SOC. SYMP., vol. 55, 1989, pages 167 - 192
FREEDMAN ET AL., BIOCHEM. SOC. TRANS., vol. 16, 1988, pages 96 - 9
FREEDMAN, TRENDS BIOCHEM. SCI., vol. 9, 1984, pages 438 - 41
FUTCHER, YEAST, vol. 4, 1988, pages 27
FUTCHER; COX, J. BACTERIOL., vol. 157, 1984, pages 283
GIETZ; SUGINO, GENE, vol. 74, 1988, pages 527 - 534
GILBERT, BIOCHEMISTRY, vol. 28, 1989, pages 7298 - 7305
GLOVER; LINDQUIST, CELL., vol. 94, 1998, pages 73 - 82
GRAINGE ET AL., J. MOL. BIOL., vol. 314, 2001, pages 717
GUENTHER R ET AL: "Functional replacement of the Saccharomyces cerevisiae Trg1/Pdi1 protein by members of the mammalian protein disulfide isomerase family", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOCHEMICAL BIOLOGISTS, BIRMINGHAM,, US, vol. 268, no. 11, 15 April 1993 (1993-04-15), pages 7728 - 7732, XP002223364, ISSN: 0021-9258 *
HARRIS; AISEN: "Physical Bioinorganic Chemistry", vol. 5, 1991, VCH, article "Iron carriers and iron proteins"
HARTL, NATURE, vol. 381, 1996, pages 571 - 580
HARTLEY; DONELSON, NATURE, vol. 286, 1980, pages 860
HAWKINS; FREEDMAN, BIOCHEM. J., vol. 275, 1990, pages 335 - 339
HAYANO ET AL., FEBS LETTERS, vol. 377, 1995, pages 505 - 511
HAYANO ET AL., FEBSLETTERS, vol. 377, 1995, pages 505 - 511
HILLSON ET AL., METHODS ENZYMOL., vol. 107, 1984, pages 281 - 292
HJELMQVIST ET AL., GENOME BIOLOGY, vol. 3, no. 6, 2002
HJELMQVIST LARS ET AL: "ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins.", GENOME BIOLOGY 2002, vol. 3, no. 6, 2002, pages RESEARCH0027.1 - 0027.16, XP002370509, ISSN: 1465-6914 *
HOHEISEL, BIOTECHNIQUES, vol. 17, 1994, pages 456 - 460
HOLLENBERG, CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOBIOLOGY, vol. 96, 1982, pages 119
IRIE ET AL., GENE, vol. 108, no. 1, 1991, pages 139 - 144
IRIE ET AL., MOL. GEN. GENET., vol. 225, no. 2, 1991, pages 257 - 265
ISOYAMA ET AL., J. BIOL. CHEM., vol. 276, no. 24, 2001, pages 21863 - 21869
ITO ET AL., J. BACTERIOL., vol. 153, 1983, pages 163
JAYARAM, PROC. NATL. ACAD. SCI. U.S.A., vol. 82, 1985, pages 5875
KASKA ET AL., BIOCHEM. J, vol. 268, 1990, pages 63 - 68
KERRY-WILLIAMS ET AL., YEAST, vol. 14, 1998, pages 161 - 169
KHAN ET AL., INT. J. BIOL. MACROMOL., vol. 30, no. 3-4, 2002, pages 171 - 8
KIKUCHI, CELL, vol. 35, 1983, pages 487
KIM ET AL., PROC. NATL. ACAD. SCI. USA., vol. 95, 1998, pages 12860 - 12865
KUNKEL ET AL., METHODS ENZYMOL., vol. 154, 1987, pages 367 - 382
LAMBERT ET AL., PROC. NATL. ACAD. SCI. USA, vol. 98, 2001, pages 4652 - 4657
LEE, BIOTECHNIQUES, vol. 12, 1992, pages 677
LIVINGSTON; HAHNE, PROC. NATL. ACAD. SCI. USA, vol. 76, 1979, pages 3727
LUNDSTROM; HOLMGREN, J. BIOL. CHEM., vol. 265, 1990, pages 9114 - 9120
MASON ET AL., BIOCHEM. J., vol. 330, no. 1, 1998, pages 35
MASON ET AL., BIOCHEMISTRY, vol. 32, 1993, pages 5472
MASON ET AL., BIOCHEMISTRY, vol. 41, 2002, pages 9448
MASON ET AL., PROTEIN EXPR. PURIF., vol. 2, 1991, pages 214
MASON ET AL., PROTEIN EXPR. PURIF., vol. 8, 1996, pages 119
MEAD ET AL., MOL. GEN. GENET., vol. 205, 1986, pages 417 - 421
MEAD ET AL., MOLECULAR & GENERAL GENETICS, vol. 205, 1986, pages 417
MEYER-LEON ET AL., COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY, vol. 49, 1984, pages 797
MOSAMMAPARAST ET AL., J. BIOL. CHEM., vol. 277, no. 1, 2002, pages 862 - 868
MURRAY ET AL., J. MOL. BIOL., vol. 200, 1988, pages 601
NGOSUWAN ET AL., J. BIOL. CHEM., vol. 278, no. 9, 2003, pages 7034 - 7042
PAINTING ET AL., J. APPLIED BACTERIOLOGY, vol. 56, 1984, pages 331
PAREKH; WITTRUP, BIOTECHNOL. PROG., vol. 13, 1997, pages 117 - 122
PARKKONEN ET AL., BIOCHEM. J, vol. 256, 1988, pages 1005 - 1011
PEMBERTON ET AL., CURR. OPIN. CELL BIOL., vol. 10, 1998, pages 392 - 399
PIHLAJANIEMI ET AL., EMBO J., vol. 6, 1987, pages 643 - 9
PIPER; CURRAN, CURR. GENET., vol. 17, 1990, pages 119
PRASAD ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 84, 1987, pages 2189
REYNOLDS ET AL., MOL. CELL. BIOL., vol. 7, 1987, pages 3566
ROBINSON ET AL., BIOLTECHNOLOGY, vol. 12, 1994, pages 381 - 384
ROBINSON; WITTRUP, BIOTECHNOL. PROG., vol. 11, 1995, pages 171 - 177
ROSE; BROACH, METH. ENZYMOL., vol. 185, 1990, pages 234 - 279
ROSE; BROACH, METHODS ENZYMOL., vol. 185, 1990, pages 234
ROSE; BROACH, METHODS ENZYMOL., vol. 185, 1990, pages 234 - 279
ROSENBERG ET AL., NATURE, vol. 312, 1984, pages 77 - 80
ROTH; PIERCE, BIOCHEMISTRY, vol. 26, 1987, pages 4179 - 82
ROTHMAN, CELL, vol. 59, 1989, pages 591 - 601
RYAN; WENTE, CURR. OPIN. CELL BIOL., vol. 12, 2000, pages 361 - 371
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001
SCHERENS ET AL., YEAST, vol. 7, 1991, pages 185 - 193
SEEDORF; SILVER, PROC. NATL. ACAD. SCI. USA., vol. 94, 1997, pages 8590 - 8595
SENECOFF ET AL., PROC. NATL. ACAD. SCI. .US.A., vol. 82, 1985, pages 7270
SENECOFF ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 82, 1985, pages 7270
SENGUPTA ET AL., J. BACTERIOL., vol. 183, 2001, pages 2306
SHERMAN ET AL.: "Methods In Yeast Genetics, A Laboratory Manual", 1986, COLD SPRING HARBOR
SHIN ET AL., PROC NATL ACAD SCI USA, vol. 92, 1995, pages 2820
SHIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 2820
SHUSTA ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 773 - 777
SLEEP ET AL., BIOLTECHNOLOGY, vol. 9, 1991, pages 183 - 187
SLEEP ET AL., BIOTECHNOLOGY, vol. 9, 1991, pages 183
SLEEP ET AL., BIOTECHNOLOGY, vol. 9, 1991, pages 183 - 187
SLEEP ET AL., YEAST, vol. 18, 2001, pages 403
SLEEP ET AL., YEAST, vol. 18, 2001, pages 403 - 421
SMITH ET AL., SCIENCE, vol. 229, 1985, pages 1219 - 1224
SOLOVYOV ET AL., J. BIOL. CHEM., vol. 279, no. 33, 2004, pages 34095 - 34100
SOUTHERN, J. MOL. BIOL., vol. 98, 1975, pages 503
SUTTON; BROACH, MOL. CELL. BIOL., vol. 5, 1985, pages 2770
TESTA: "Proteins of iron metabolism", 2002, CRC PRESS
THOMPSON ET AL., NUCLEIC ACIDS RES., vol. 22, no. 22, 1994, pages 4673 - 80
TOH-E ET AL., BASIC LIFE SCI., vol. 40, 1986, pages 425
TOYN ET AL., YEAST, vol. 16, 2000, pages 553 - 560
UETA ET AL., J. BIOL. CHEM., vol. 278, no. 50, 2003, pages 50120 - 50127
VALKONEN ET AL., APPLIED ENVIRON. MICRO., vol. 69, 2003, pages 2065
VOLKERT ET AL., MICROBIOLOGICAL REVIEWS, vol. 53, 1989, pages 299
VOLKERT; BROACH, CELL, vol. 46, 1986, pages 541
WEEKE, B.: "Rocket immunoelectrophoresis", 1976
WILLIAMSON, YEAST, vol. 1, 1985, pages 1 - 14
YAMAUCHI ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 146, 1987, pages 1485 - 1492
ZEALEY ET AL., MOL. GEN. GENET., vol. 211, 1988, pages 155 - 159

Cited By (44)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8252551B2 (en) 2003-12-23 2012-08-28 Novozymes Biopharma Dk A/S 2-micron family plasmid and use thereof
WO2006136831A3 (en) * 2005-06-22 2007-02-15 Delta Biotechnology Ltd Gene expression technique
WO2006136831A2 (en) * 2005-06-22 2006-12-28 Novozymes Delta Limited Gene expression technique
WO2009019314A1 (en) 2007-08-08 2009-02-12 Novozymes A/S Transferrin variants and conjugates
EP2594583A1 (en) 2007-08-08 2013-05-22 Novozymes Biopharma DK A/S Transferrin variants and conugates
EP2604623A2 (en) 2007-08-08 2013-06-19 Novozymes Biopharma DK A/S Transferrin variants and conjugates
EP2304045B1 (en) * 2008-07-28 2018-05-09 evocatal GmbH Chaperone toolbox
US9315565B2 (en) 2009-02-10 2016-04-19 Ucb Pharma, S.A. Method for producing protein
WO2010092335A1 (en) * 2009-02-10 2010-08-19 Ucb Pharma S.A. Method for producing protein
US9476081B2 (en) 2009-02-10 2016-10-25 Ucb Biopharma Sprl Method for producing protein
US8828719B2 (en) 2009-02-10 2014-09-09 Ucb Pharma, S.A. Method for producing protein
US9493545B2 (en) 2009-02-11 2016-11-15 Albumedix A/S Albumin variants and conjugates
US11555061B2 (en) 2009-02-11 2023-01-17 Albumedix, Ltd Albumin variants and conjugates
WO2010092135A2 (en) 2009-02-11 2010-08-19 Novozymes Biopharma Uk Ltd. Albumin variants and conjugates
EP3243835A1 (en) 2009-02-11 2017-11-15 Albumedix A/S Albumin variants and conjugates
WO2010133668A2 (en) 2009-05-20 2010-11-25 Fh Campus Wien Eukaryotic host cell comprising an expression enhancer
EP2258854A1 (en) 2009-05-20 2010-12-08 FH Campus Wien Eukaryotic host cell comprising an expression enhancer
US10696732B2 (en) 2009-10-30 2020-06-30 Albumedix, Ltd Albumin variants
US10233228B2 (en) 2010-04-09 2019-03-19 Albumedix Ltd Albumin derivatives and variants
US10711050B2 (en) 2011-11-18 2020-07-14 Albumedix Ltd Variant serum albumin with improved half-life and other properties
US9944691B2 (en) 2012-03-16 2018-04-17 Albumedix A/S Albumin variants
US10329340B2 (en) 2012-03-16 2019-06-25 Albumedix Ltd Albumin variants
US10934341B2 (en) 2012-11-08 2021-03-02 Albumedix, Ltd. Albumin variants
US10501524B2 (en) 2012-11-08 2019-12-10 Albumedix Ltd Albumin variants
EP4311857A2 (en) 2014-04-17 2024-01-31 Boehringer Ingelheim RCV GmbH & Co KG Recombinant host cell engineered to overexpress helper proteins
WO2015158808A2 (en) 2014-04-17 2015-10-22 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell engineered to overexpress helper proteins
WO2015158800A1 (en) 2014-04-17 2015-10-22 Boehringer Ingelheim Rcv Gmbh & Co Kg Recombinant host cell for expressing proteins of interest
CN105985428A (en) * 2015-02-10 2016-10-05 许健 Procoagulant function of human recombinant blood coagulation factor XIII
US10633428B2 (en) 2015-08-20 2020-04-28 Albumedix Ltd Albumin variants and conjugates
US12116400B2 (en) 2015-08-20 2024-10-15 Sartorius Albumedix Limited Albumin variants and conjugates
US10023618B2 (en) 2015-12-22 2018-07-17 Albumedix A/S Protein expression strains
WO2017112847A1 (en) 2015-12-22 2017-06-29 Albumedix A/S Improved protein expression strains
WO2018234349A1 (en) 2017-06-20 2018-12-27 Albumedix Ltd Improved protein expression strains
EP4026908A1 (en) 2017-06-20 2022-07-13 Albumedix Ltd Improved protein expression strains
US11396670B2 (en) 2017-06-20 2022-07-26 Albumedix Limited Protein expression strains
US11130979B2 (en) 2017-06-20 2021-09-28 Albumedix Ltd Protein expression strains
US11667922B2 (en) 2018-03-26 2023-06-06 Novozymes A/S Fungal chaperone proteins
WO2019185535A1 (en) * 2018-03-26 2019-10-03 Novozymes A/S Fungal chaperone proteins
EP3716274A1 (en) 2019-03-29 2020-09-30 Ajinomoto Co., Inc. Control device, control method, computer program, and method for producing organic compound
EP4194560A1 (en) * 2021-12-08 2023-06-14 European Molecular Biology Laboratory Improved production of secreted proteins in fungal cells
WO2023104896A1 (en) * 2021-12-08 2023-06-15 Lesaffre Et Compagnie Improved production of secreted proteins in fungal cells
WO2023104902A1 (en) * 2021-12-08 2023-06-15 Lesaffre Et Compagnie Improved production of secreted proteins in yeast cells
CN118460394A (en) * 2024-07-12 2024-08-09 通化安睿特生物制药股份有限公司 Engineering bacterium for preparing transferrin and application thereof
CN118460394B (en) * 2024-07-12 2024-09-24 通化安睿特生物制药股份有限公司 Engineering bacterium for preparing transferrin and application thereof

Also Published As

Publication number Publication date
US20080193977A1 (en) 2008-08-14
US20120231505A1 (en) 2012-09-13
US9057061B2 (en) 2015-06-16
JP5631533B2 (en) 2014-11-26
EP1831375A1 (en) 2007-09-12
AU2005317828A1 (en) 2006-06-29
EP2330200B1 (en) 2017-04-05
US20160186192A1 (en) 2016-06-30
EP1831375B1 (en) 2014-07-16
JP2008525007A (en) 2008-07-17
DK2330200T3 (en) 2017-07-24
US8969064B2 (en) 2015-03-03
EP2330200A2 (en) 2011-06-08
EP2330200A3 (en) 2011-12-28

Similar Documents

Publication Publication Date Title
US9057061B2 (en) Gene expression technique
US20180105818A1 (en) Gene expression technique
CA2551496C (en) 2-micron family plasmid and use thereof
JP5781986B2 (en) Gene expression technology

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LC LK LR LS LT LU LV LY MA MD MG MK MN MW MX MZ NA NG NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU LV MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2005317828

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2007547668

Country of ref document: JP

ENP Entry into the national phase

Ref document number: 2005317828

Country of ref document: AU

Date of ref document: 20051223

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2005823505

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2005317828

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 11722539

Country of ref document: US

WWE Wipo information: entry into national phase

Ref document number: 2783/CHENP/2007

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 200580048615.0

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 2005823505

Country of ref document: EP