WO2006136831A2 - Gene expression technique - Google Patents

Gene expression technique Download PDF

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Publication number
WO2006136831A2
WO2006136831A2 PCT/GB2006/002289 GB2006002289W WO2006136831A2 WO 2006136831 A2 WO2006136831 A2 WO 2006136831A2 GB 2006002289 W GB2006002289 W GB 2006002289W WO 2006136831 A2 WO2006136831 A2 WO 2006136831A2
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WO
WIPO (PCT)
Prior art keywords
ssal
ssel
sse2
ssbl
ssb2
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PCT/GB2006/002289
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French (fr)
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WO2006136831A3 (en
Inventor
Thomas Payne
Darrell Sleep
Christopher John Arthur Finnis
Leslie Robert Evans
Original Assignee
Novozymes Delta Limited
University Of Nottingham
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Application filed by Novozymes Delta Limited, University Of Nottingham filed Critical Novozymes Delta Limited
Priority to CN200680022631.7A priority Critical patent/CN101203610B/en
Priority to EP06755593A priority patent/EP1896591A2/en
Priority to JP2008517592A priority patent/JP5107910B2/en
Priority to US11/993,335 priority patent/US20110020865A1/en
Priority to AU2006260739A priority patent/AU2006260739B2/en
Publication of WO2006136831A2 publication Critical patent/WO2006136831A2/en
Publication of WO2006136831A3 publication Critical patent/WO2006136831A3/en
Priority to US13/784,095 priority patent/US20130244277A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present application relates to gene expression techniques.
  • a key parameter in the development of a commercially viable process for the production of a recombinant protein is the yield of the product from the host organism.
  • Factors that influence the yield of a particular heterologous protein are complex and include the biochemical and biophysical properties of the protein itself; its influence on, and modification of, the host's own cellular functions; and the choice and deployment of those sequences that are necessary for efficient transcription, translation, secretion (if required) and plasmid stability.
  • helper proteins proteins that are over-expressed in a (non-publicly available) S. cerevisiae that possesses increased production of a protein product of choice, such as a recombinant protein.
  • helper proteins have all, individually, been previously identified.
  • helper proteins there is nothing in the art to suggest that their over-expression would aid in the increased production of a recombinant heterologous protein product of choice.
  • the (as yet unpublished) art has recognised that their over-expression can aid in increasing the production of a recombinant heterologous protein product of choice (see PCT/GB2004/005462).
  • PCT/GB2004/005462 there is nothing in the art to suggest that the combined and simultaneous over-expression of such helper proteins would further enhance the production of a protein product of choice.
  • the present invention provides a host cell suitable for enhanced production of a protein product of choice wherein the host cell is genetically modified to cause over-expression of one or more of the identified helper proteins.
  • the present invention provides a host cell that is suitable for enhanced production of a protein product of choice characterised in that the host cell comprises a first gene encoding a first helper protein as defined herein, or a variant thereof, and a second gene encoding a desired protein product of choice, wherein the host cell is genetically modified to cause over-expression of the first helper, protein, and-
  • the host cell is not genetically modified to cause over-expression of a further helper protein that is different from the first helper protein and is selected from the group consisting of AHAl, CCT2, CCTS, CCT4, CCT5, CCT6, CCTl, CCT8, CNSl, CPR3, CPR6, EROl, EUGl, FMOl, HCHl, HSPlO, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, JEMl, MDJl, MDJ2, MPDl, MPD2, PDIl, PFDl, ABCl, APJl,
  • a further helper protein that is different from the first helper protein and is selected from the group consisting of AHAl, CCT2, CCTS, CCT4, CCT5, CCT6, CCTl, CCT8, CNSl, CPR3, CPR6, EROl, EUGl, FMOl, HCHl, HSPlO, HSP12, HSP104,
  • the thus over-expressed first helper protein may be any helper protein defined below.
  • the over-expressed first helper protein may be a DnaJ-like protein (such as JEMl), an Hsp70 family member protein (such as LHSl) or SILl 5 or a variant of any of these.
  • Over-expression of the first helper protein may be achieved by any suitable means of genetic modification known in the art. Suitable examples of such approaches for genetic modification are discussed in more detail below.
  • the host cell may or may not comprise a recombinant copy, such as a plasmid encoded copy, or a chromosomally integrated recombinant copy, of a gene encoding the further helper protein as defined in (b) above.
  • the first helper protein may be the only helper protein that is over- expressed by the host cell.
  • the invention provides a host cell that is suitable, for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over-expression of a helper protein selected from the list comprising SCJl, FKB2, SSEl, ERV2, DERl 5 DER3, HRD3, UBC7 and D0A4.
  • the host cell may or may not be genetically modified to cause over- expression of two or more helper proteins, at least one of which is a helper protein selected from the list comprising SCJl, FKB2, SSEl, ERV2, DERI, DER3, HRD3, UBC7 and DOA4.
  • at least one other helper may or may not be selected from the list comprising -
  • Hsp70 family member protein such as LHSl
  • SCJl such as SCJl
  • KAR2 SILl
  • SILl note that, SILl has previously been referred to as SLSl
  • FKB2 SSAl 5 SSA2, SSA3, SSA4, SSEl, SSE2, SSBl 5 SSB2, ECMlO 5 MDJl and MDJ2.
  • proteins involved in the formation of disulphide bonds in other proteins selected from ERO 1 , ERV2 5 EUGl , MPD 1 , MPD2, EPS 1 and PDIl;
  • proteins involved in protein degradation selected from DERI, DER3, HRD3, UBC7 and D0A4;
  • the host cell may or may not be genetically modified to cause over- expression of two or more helper proteins selected from a DnaJ-like protein (such as JEMl), an Hsp70 family protein (such as LHSl) and SJXl.
  • the host cell according to may or may not be genetically modified to cause over- expression of-
  • the host may or may not be genetically modified to cause over-expression of three or more helper proteins, wherein the three or more helper proteins comprise a DnaJ-like protein, an Hsp70 family protein and SILl, for example JEMl 5 LHSl and SILl.
  • helper proteins comprise a DnaJ-like protein, an Hsp70 family protein and SILl, for example JEMl 5 LHSl and SILl.
  • the Hsp70 family protein may or may not be a protein that localises to the lumen of the ER.
  • the Hsp70 family protein may or may not be a prokaryotic Hsp70 family protein.
  • the Hsp70 family protein may or may not be a eukaryotic Hsp70 family protein.
  • the Hsp70 family protein may or may not be LHSl 5 KAR2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 or ECMlO, such as from yeast, for example, from S. cerevisiae.
  • LHSl may or may not be a preferred Hsp70 family protein for use in the present invention.
  • Other Hsp70 family proteins for use in the present invention may or may not include a mammalian BiP
  • HSP72 HSP72
  • HSC70 HSP73
  • mtp70 a mammalian GRP170 (such as the protein described by Lin et al (1993) MoI. Biol Cell 4,
  • HSP70 protein such as a protein as reviewed by Ohtsuka and Hata. (2000) International Journal of Hyperthermia 16, 231; Gething and
  • Gallus g ⁇ llus HSP70 protein such as the protein defined by accession number AAO44921 (Mazzi et al (2003) Genet. MoI. Biol. 26, 275-281), a Nicotiana tabacum luminal binding protein (BiP), such as the protein defined by accession number CAA42661 (Denecke et al (1991) Plant Cell 3, 1025), a Paramecium caudatum HSP70 protein, such as the protein defined by accession number
  • aHordeum vulgar e HSP70 protein such as a subsp. vulgare HSP70 protein accession number, such as the protein defined by AAA62325 (Chen et al (1994) Plant Physiol. 106, 815), an
  • Chlamydia trachomatis A/HAR-13 chaperone protein dnaK Heat shock protein
  • HSP70 Heat shock 70 kDa protein
  • HSP70 Heat shock 70 kDa protein
  • accession number Q3KLV7 Carlson et al (2005) Infect. Immun. 73, 6407
  • Pongo pygmaeus hsp70 protein such as the protein defined by accession number
  • Streptococcus pneumoniae HSP70 protein such as the protein defined by accession number AAB39221
  • Mus musculus HSP70 protein such as the protein defined by accession number AAC84169 (Xie et al (2003)
  • Bacillus subtilis HSP70 protein such as the protein defined by accession number BAA12464 (Mizuno et al (1996) Microbiology
  • LHSl may or may not be taken to be, by extension, a reference to an equivalent Hsp70 family protein, such as an Hsp70 family protein as defined in this paragraph.
  • Hsp70 family proteins may have an activity equivalent to LHSl, when co-expressed with one or both of JEMl and SlLl, for example in the manner as set out in the present examples.
  • a host cell of the present invention when genetically modified to cause simultaneous over-expression of a preferred Hsp70 family protein with one or both of JEMl and SILl, will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl 5 the increase being compared to the to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been geneticalfy modified to cause overexpression of any of LHSl, JEMl or SILl.
  • substantially the same increase in the production of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl (the increased being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SJXl).
  • substantially the same reduction of fragmentation of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SJXl). DnaJ-like proteins are reviewed in Walsh et al, 2004, EMBO reports, 5, 567-571.
  • the DnaJ-like protein typically comprises a J-domain as defined in Walsh et a ⁇ , 2004, op. cit. the contents of which are incorporated herein by reference.
  • the DnaJ-like protein may or may not be a prokaryotic DnaJ-like protein.
  • the DnaJ- like protein may or may not be a eukaryotic DnaJ-like protein.
  • the DnaJ-like protein may or may not be any one of the yeast DnaJ proteins such as a protein selected from JEMl 5 MDJl, MDJ2, SEC63, YDJl 5 XDJl 5 APJl 5 SISl 5 DJPl 5 ZUOl 5 SWA2, JJJl 5 JJJ2, JJJ3, CAJl 5 CWC23, PAMlS 5 JACl, JIDl 5 SCJl, HLJl and ERJ5.
  • the DnaJ-like protein may or may not be a protein that localises to the ER 5 such as JEMl 5 SCJl, HLJl 5 SEC63 or ERJ5, and may or may not be a protein that localises to the ER membrane.
  • the DnaJ-like protein may or may not be a protein that localises to the cytoplasm of the host cell, such as YDJl, XDJl, APJl, SISl 5 DJPl, ZUOl, SWA2, JJJl 5 JJJ2 or JJJ3.
  • the DnaJ-like protein may or may not be a protein that localises to the nucleoplasm of the host cell, such as CAJl or CWC23.
  • the DnaJ-like protein may or may not be a protein that localises to the mitochondria of the host cell, such as MDJl, MDJ2, PAM18, JACl or JlDl.
  • the DnaJ-like protein is typically not SCJl.
  • JEMl may or may not be a preferred DnaJ-like protein for use in the present invention.
  • Other DnaJ-like proteins may or may not include the following proteins or proteins families, or fragments or variants thereof -
  • a mammalian Erdj3 such as HEDJ/Scjlp, Shen and Hendershot (2005) MoI. Biol. Cell. 16, 40
  • a mammalian Erdj4 such as described in Shen et al (2002) J Biol. Chem. 277, 15947
  • a mammalian Erdj5 such as described in Cunnea et al (2003) J Biol. Chem. 278, 1059
  • a Gallus gallus DnaJ homolog subfamily B member 11 precursor such as the ER-associated dnaJ protein 3 ErJ3, the ER-associated Hsp40 co- chaperone (hDj9, or the PWPl -interacting protein 4, such as defined by accession number XP_422682;
  • Nicotiana tabacum DnaJ homolog such as the protein defined by accession number BAC53943;
  • Arabidopsis thaliana DnaJ homolog such as the protein defined by accession number AAB49030 (Zhou et al (1999) Plant Physiol. 121,
  • dnaJ Chlamydia trachomatis A/HAR-13 Chaperone protein dnaJ, such as the protein defined by accession number YP_328153 (Carlson et al (2005) Infect. Immun. 73, 6407); • a Pongo pygmaeus DnaJ homolog subfamily B member 9, such as the protein defined by accession number Q5R9A4;
  • a Haemophilus influenzae Rd KW20 Dna-J like membrane chaperone protein such as the protein defined by accession number NP_438440 (Fleischmann et al (1995) Science 269, 496); • a Escherichia coli DnaJ protein, such as the protein defined by accession number AAA00009 (Ohki et al (1986) J. Biol. Chem. 261, 1778);
  • a Escherichia coli DnaJ-like protein such as the protein defined by accession number BAB96590 (Musso et al (1977) Proc. Natl. Acad. Sd. U.S.A. 74, 106); • a Streptococcus pneumoniae DnaJ protein, such as the protein defined by accession number AAB39222;
  • a Mus musculus DnaJ homolog such as a subfamily B member 6 (Heat shock protein J2) (HSJ-2) (MRJ) (mDj4) , such as the protein defined by accession number XP_987742; • a Bacillus subtilis DnaJ protein, such as the protein defined by accession number BAA12465 (Mizuno et al (1996) Microbiology (Reading, Engl.) 142, 3103); and • a plant Sorghum bicolour DNAJ domain protein, such as the protein defined by accession number ABF48023.
  • a Mus musculus DnaJ homolog such as a subfamily B member 6 (Heat shock protein J2) (HSJ-2) (MRJ) (mDj4) , such as the protein defined by accession number XP_987742
  • a Bacillus subtilis DnaJ protein such as the protein defined by accession number BAA12465 (Mizuno et al (1996) Microbiology (Read
  • JEMl may or may not be taken to be, by extension, a reference to an equivalent DnaJ-like protein, such as a DnaJ-like protein as defined in the above paragraph.
  • a host cell of the present invention when genetically modified to cause simultaneous over-expression of a preferred DnaJ- like protein with one or both of LHSl and SILl, will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of JEMl with one or both of LHSl and SILl, the increase being compared to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl.
  • substantially the same increase in the production of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase, in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of JEMl with one or both of LHSl and SILl (the increase being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
  • substantially the same reduction of fragmentation of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of JEMl with one or both of LHS 1 and SILl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
  • the host cell that is genetically modified to cause over-expression of two or more, such as at least three, helper proteins selected from a DnaJ-like protein, an Hsp70 family protein and SILl may or may not be further genetically modified to cause over-expression of at least one, two, three, four, five, six or seven proteins involved in the formation of disulphide bonds in other proteins selected from the group consisting of EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
  • PDIl may or may not be preferred.
  • the invention provides a host cell suitable for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over-expression of three or more helper proteins, wherein the three or more' helper proteins are selected from the list comprising —
  • Hs ⁇ 70 family member protein (such as LHSl) 5 SCJl, KAR2, SILl, FKB2, SSAl, SSAV SSA3, SSA4, SSEl, SSE2, SSBl 5 SSB2, ⁇ ECMl O 5 MDJl and MDJ2.
  • proteins involved in the formation of disulphide bonds in other proteins selected from EROl, ERV2, EUGl 5 MPDl, MPD2, EPSl and PDIl;
  • proteins involved in protein degradation selected from DERI, ⁇ DER3, HRD3, UBC7 and D0A4;
  • the three or more helper proteins may or may not comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the chaperones selected from the group consisting of JEMl 5 an Hsp70 family member protein (such as LHSl) 5 SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl and MDJ2.
  • the three or more helper proteins may or may not comprise at least one, two, three, four, five, six or seven proteins involved in the formation of disulphide bonds in other proteins selected from the group consisting of EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
  • the three or more helper proteins may or may not comprise at least one, two, three, four or five of the proteins involved in protein degradation selected from DERI, DER3, HRD3, UBC7 and DOA4.
  • the host cell may or may not comprise a polynucleotide sequence that encodes a protein product of choice.
  • the host cell comprises a polynucleotide sequence that encodes a protein product of choice.
  • the protein product of choice may or may not be a protein that is naturally produced by the host cell or may or may not be a heterologous protein.
  • a heterologous protein is a protein that is not naturally encoded by the host cell.
  • the polynucleotide sequence that encodes the protein product of choice may or may not be an endogenous polynucleotide sequence or (in particular, where the protein product of choice is a heterologous protein) the polynucleotide sequence that encodes the protein product of choice may or may not be an exogenous polynucleotide, and the exogenous polynucleotide may or may not be integrated into the chromosome of the host cell or present in the host cell as part of a replicable vector, such as a plasmid.
  • the present invention also contemplates the production of host cells suitable for enhanced production of a protein product of choice, into which an appropriate polynucleotide sequence, encoding the protein product of choice, can be later introduced. Therefore, in another embodiment, the host cell does not comprise a polynucleotide sequence that encodes a protein product of choice. Suitable host cells are discussed below.
  • enhanced production we include the meaning that the level of production of protein product of choice is greater in a cultured population of the genetically modified host cell than in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins.
  • the measurement can be made under culture conditions that are standard for the growth of the host cell that is being used.
  • the production of the protein product of choice in a cultured population of the genetically modified host cell of the invention be greater than, typically at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (i.e. 1.1-fold), 20% (i.e. 1.2-fold), 30% (Le. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. l:8-fold), 90% (i.e. 1.9-fold), 100% (i.e.
  • the production of the protein product of choice in a cultured population of the genetically modified host cell of the invention may be up to 10% (Le. 1.1-fold), 2O°/o (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (Le. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9-fold), 100% (Le.
  • the protein product of choice may be produced in a cultured population of the genetically modified host cell of the invention to produce a culture containing at least 0.001 g.L “1 , such as at least 0.01 g.L “1 , at least 0.1 g.L “1 , 1 g.L “1 , 2 g.L “1 , 3 g.1/ 1 , 4 g.1/ 1 , 5 g.1/ 1 , 6 g.1/ 1 , 7 g.1/ 1 , 8 g.1/ 1 , 9 g.L/ 1 , 10 g.L- 1 , 20 g.1/ 1 , 30 g.L/ 1 , 40 g.L “1 , 50 g.L “1 , 60 g.L/ 1 , 70 g.L/ 1 , 80 g.L 4 , 90 g.L “1 , or 100 g.L “1 of the protein product of choice.
  • the protein product of choice may be produced in a cultured population of the genetically modified host cell of the invention to produce a culture containing up to 0.01 g.L “1 , 0.1 g.L “1 , 1 g.L “1 , 2 g.L “1 , 3 g.L “1 , 4 g.L “1 , 5 g.L “1 , 6 g.1/ 1 , 7 g.1/ 1 , 8 g.L “1 , 9 g.L “1 , 10 g.L “1 , 20 g.L “1 , 30 g.L “1 , 40 g.1/ 1 , 50 g.L “1 , 60 g.1/ 1 , 70 g.L “1 , 80 g.L “1 , 90 g.L '1 , 100 g.L “1 or 200 g.L “1 of the protein product of choice.
  • enhanced production we also include the meaning that the level of activity of the protein product of choice that is produced by the host cell is greater in a cultured population of the genetically modified host cell than in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins.
  • the nature of the activity will depend on the identity of the protein product of choice and may, for example, be a measurement of the catalytic activity of the protein upon a substrate or the binding properties of rtie protein to a ligand.
  • the measurement of protein activity can be made under culture conditions that are standard for the growth of the host cell that is being used or following isolation of the. protein from the culture medium. Ln either case, the comparison should be made on the basis of activity per unit volume of culture or protein recovered . therefrom. The comparison may, or may not, be normalised to account for differences in the cell growth of the two cultured populations, as compared.
  • the activity of the protein product of choice that is produced in a cultured population of the genetically modified host cell of the invention may be greater than, typically at least 10% (i.e. 1.1-fold), 20% (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7-fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9-fold), 100% (i.e.
  • the activity of the protein product of choice in a cultured population of the genetically modified host cell of the invention may be up to 10% (i.e. 1.1- fold), 20% (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5- fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9- fold), 100% (Le.
  • enhanced production we include the additional or alternative meaning that the level of degradation of the protein product of choice is reduced when produced by a cultured population of the genetically modified host cell of the present invention compared to the level of degradation of the protein product of choice when produced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention.
  • the level of protein degradation can be determined by quantification of fragments of the protein product of choice relative to the total of the protein product of choice, for example when by analysis of SDS-PAGE using densitometry. When expressed as a percentage of detected protein product fragments. relative to total protein product levels detected (i.e.
  • the percentage of detected protein product fragments when produced by a cultured population of the genetically modified host cell of the present invention may be, or be less than, 99%, 98%, 97%, 96%, 05%, 04%, 03%, 92%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, such as up to 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, such as up to 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 85%
  • enhanced production we include the additional or alternative meaning that the level of post-translational modification of the protein product of choice is increased or reduced when produced by a cultured population of the genetically modified host cell of the present invention compared to the level of post- translational modification of the protein product of choice when produced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention.
  • altered production i.e.
  • level of post-translational modification may be an alteration in the level of proteolytic cleavage, hexosylation (for example mannosylation), glycosylation, phosphorylation, phosphopantetheinylation, carbamylation, carboxylation (such as ⁇ -carboxylation), sialation, sulphonation, hydroxylation, prenylation, isoprenylation, acylation, ubiquitination, lipoylation, biotinylation, glycylation, glutamylation, methylation, . alkylation, acetylation, formylation, selenation, disulphide bond formation or oligomerisation of the protein product of choice .
  • the level of post-translational modification of the protein product of choice can be determined by methods well known in the art, such as by mass spectrometry techniques (for example, see Larsen et al, 2006, BioTechniques, 40, 790-798) well known in the art.
  • enhanced production we include the additional or alternative meaning that the level of stress experienced by a cell that is being cultured to produce the protein product of choice is reduced, compared to the level of stress experienced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention.
  • enhanced production can include the additional or alternative meaning that the unfolded protein response is reduced in a host cell.
  • the level of stress, and the level of the unfolded protein response can be measured by determination of the proportion of HACl 1 to total HACl transcript levels.
  • Total HACl transcript levels are the sum of HACl 1 transcript levels and unspliced HACl (HACl”) transcript levels in a cell.
  • Helper proteins suitable for achieving this effect may include Hsp70 family proteins (such as LHSl) and DnaJ-like proteins (such as JEMl) and combinations of other helper proteins such as disclosed in the present application.
  • any "protein product of choice” can be produced.
  • the identity of preferred embodiments of the "protein product of choice” is discussed further below.
  • the host cell is genetically modified to cause over-expression of one or more of the helper proteins.
  • over-expression in the context of helper proteins, we mean that the measurable level of mRNA encoding the one or more helper proteins, and/or the measurable level of the one or more helper proteins themselves, and/or the measurable level of the helper protein activity, is greater than the measurable level in a host cell that has not been genetically modified.
  • the measurement will be made under culture conditions that are standard for the growth of the host cell that is being used. Standard conditions for yeast cell growth are discussed, for example, in WO 96/37515, WO 00/44772 and WO 99/00504, the contents of which are incorporated herein by reference.
  • the host cell may or may not be genetically modified to cause a level of expression of one or more of the helper proteins that is at least a 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or more, of the unmodified level of expression of one or more of the helper proteins.
  • the host cell may or may not be genetically modified to cause a level of expression of one or more of the helper proteins that is up to 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold of the unmodified -type level of expression of one or more of the helper proteins.
  • the host cell may be genetically modified to cause a level of expression of one or more of the helper proteins that is between 1- to 30-fold, such as about 2- to 25-fold, of the unmodified-type level of expression of one or more of the helper proteins.
  • the host cell may or may not be genetically modified to cause over-expression of one or more of the helper proteins by the introduction of one or more recombinant copies of one or more polynucleotides that each comprise a region (the "coding region”, or “open reading frame”, which can be abbreviated to "ORF") that encodes one or more helper proteins.
  • a copy of the polynucleotide may or may not be introduced into the chromosome of the host cell and/or may or may not be encoded by a plasmid or other vector that is used to transform the host cell.
  • the polynucleotide may or may not comprise some or all of the regulatory sequences necessary to cause transcription and/or translation of the ORP of the polynucleotide.
  • Regulatory sequences necessary to cause transcription and/or translation of the ORF of the polynucleotide include sequences that modulate (i.e., promotes or reduces, typically promotes) 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 be about, or up to, 5bp, lObp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 60bp, 70bp, 80bp, 90bp, lOObp, 120bp, 140bp, 160bp, 180bp, 200bp, 220bp, 240bp, 260bp, 280bp, 300bp, 35Obp, 400b ⁇ , 450bp, 500b ⁇ , 550bp, 60,0bp, 650bp, 700b ⁇ , 750bp, 800bp, 850b ⁇ , 900bp, 950b ⁇ , lOOObp, HOObp, 1200bp, 1300bp, 1400bp, 1500bp or greater, in length. '
  • Such non-coding regions and regulatory regions are not restricted to the native non-coding regions and/or regulatory regions naturally associated with the ORF.
  • suitable promoters for S. cerevisiae include those associated with the PGKl gene, GALl or GALlO genes, TEFl, TEF2, PYKl, PMAl, CYCl, PH05, TRPl, ADHl, ADH2, the genes for glyceraldehyde-3 -phosphate dehydrogenase (for example, TDHl, TDH2 or
  • TDH3 hexokinase (for example, HXKl or HXK2), pyruvate decarboxylase (for example, PDCl, PDC5 or PDC6), phosphofructokinase (for example, PFKl or PFKl), triose phosphate isomerase (for example, TPIl), phosphoglucose isomerase (for example, PGIl), glucokinase (for example, GLKl), ⁇ -mating factor pheromone (for example, MFa-I or MFa-2), a-mating factor pheromone (for example, MFAl or MFA2), PRBl, PRAl, GPDl, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g.
  • the promoter of EP-A-258 067) where multiple ORFs are to be expressed, a different promoter may or may not be chosen for each ORF.
  • the skilled person can readily determine appropriate combinations of promoters. For example, the promoters from the ADHl, PGKl, TDHl and TEFl genes are used in combination to recombinantly over-express four helper proteins in Example 3 below.
  • Suitable transcription termination signals are well known in the art. Where the host cell is eukaryotic, the transcription termination signal is preferably derived from the 3' flanking sequence of a eukaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different. Li that case, and where the host is a yeast, preferably S. cerevisiae, then the termination signal of the & cerevisiae ADHl, ADH2, CYCl, or PGKl genes are preferred.
  • promoter and open reading frame may 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, and visa versa.
  • a suitable regulatory sequences in yeast such as Saccharomyces cerevisiae, includes: a yeast promoter (e.g. the Saccharomyces cerevisiae PRBl promoter), as taught in EP 431 880; and a transcription terminator, preferably the terminator from Saccharomyces ADHl, as taught in EP 60 057.
  • Other suitable regulatory sequences are given in. the examples, and include TEFl, PGKl and TDHl promoters.
  • the non-coding region may incorporate more than one DNA sequence encoding a translational stop codon, such as UAA 5 UAG or UGA 5 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 polynucleotide incorporates at least two translation stop codons.
  • 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 ORF in its intended manner.
  • a regulatory region "operably linked" to an ORF is positioned in such a way that the regulatory region is able to influence transcription and/or translation of the ORF in the intended manner, under conditions compatible with the regulatory sequence.
  • the polynucleotide may or may not be formed in such a manner that it can take advantage of endogenous regulatory sequences within the chromosome or plasmid to cause transcription and/or translation of the coding region of the polynucleotide.
  • endogenous regulatory sequences within the chromosome or plasmid to cause transcription and/or translation of the coding region of the polynucleotide.
  • promoterless constructs is well known in the art as a way of allowing an endogenous promoter sequence to drive the expression of a recombinantly-introduced polynucleotide coding region.
  • the host cell may or may not comprise endogenous copies of genes encoding one or more of the helper proteins. Therefore, this invention also contemplates genetic modifications to the host cell that cause increased steady state levels of mRNA molecules encoding one or more helper proteins and/or increased steady state levels of one or more helper proteins.
  • the endogenous promoter in the gene of an endogenously encoded helper protein can be replaced by a promoter that causes greater levels of expression of the helper protein under culture conditions.
  • genetic modifications can be made to cis or trans regulators of the gene of an endogenously encoded helper protein, so as to increase the expression of the helper protein under culture conditions.
  • the polynucleotide region that encodes a genetically encoded repressor of a gene of an endogenously encoded helper protein could be genetically modified to reduce or prevent repression of the endogenous helper protein gene.
  • helper protein or protein product of choice can involve transient expression techniques known in the art. For example, suitable techniques are disclosed in Chen et al, 1997, Nucleic Acids Research, 25, 4416-4418 and in Behr et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 6982-6986.
  • Suitable techniques include -
  • the host cell comprises a first gene encoding a protein product of choice, and a second gene encoding a first helper protein, then for example,
  • the first gene may be a gene as defined in (i) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (ii) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the first and second genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the second gene);
  • the first gene may be a gene as defined in (iii) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iv) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; or • the first gene may be a gene as defined in (v) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above.
  • the host cell comprises a first gene encoding a protein product of choice, and a second gene encoding a first helper protein and a third gene encoding a second helper protein, then for example,
  • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
  • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene 4 as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above;
  • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the first and third genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the third gene);
  • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where the first, second and third genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the second gene, the first gene may or may not be introduced on the same plasmid or vector as the third gene and the second gene may or may not be introduced on the same plasmid or vector as the third gene); • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above;
  • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (i) above,- and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
  • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
  • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as. defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above;
  • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i) 5 (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
  • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
  • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; or • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iv) or (v) above.
  • helper proteins in a host cell, at least one helper protein may or may not be over-expressed by the introduction of an appropriate recombinant polynucleotide sequence as discussed above, whereas at least one other helper protein may or may not be over-expressed by a genetic modification to the host cell to cause over-expression of the helper protein from the endogenous gene that encodes it.
  • helper proteins proteins that are over-expressed in a S. cerevisiae strain identified as possessing increased production of a recombinant protein. These over-expressed helper proteins have all, individually, been previously identified.
  • helper proteins identified include proteins that can be categorised as follows -
  • HACl encoded by a spliced or unspliced polynucleotide
  • chaperones The class of proteins known as chaperones have been defined by Hartl (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.
  • chaperones of interest can be broadly split into the following three functional sub-groups -
  • ER luminal localised chaperones involved in "protein folding” include DnaJ-like proteins (such as JEMl), Hsp70 family member proteins (such as LHSl), SCJl, KAR2, SILl and FKB2. A detailed description of these proteins and their genes is given separately below.
  • the host cell may or may not be genetically modified to cause over-expression of one, or more, of the above ER luminal localised chaperones.
  • SCJl may or may not be over-expressed.
  • FKB2 may or may not be over-expressed.
  • the host cell may or may not be genetically modified to cause over-expression of two of the above ER luminal localised chaperones.
  • one of the folio wing combinations may or may not be chosen -
  • a DnaJ-like proteins such as JEMl
  • an Hsp70 family member protein such as LHS 1
  • SCJl a DnaJ-like proteins
  • KAR2 a DnaJ-like proteins
  • FKB2 a DnaJ-like proteins
  • Hsp70 family member protein (such as LHS 1) in combination with one of SCJl, KAR2, SILl or FKB2;
  • the host cell may or may not be genetically modified to cause over-expression of three of the above ER luminal localised chaperones.
  • the following combinations may or may not be chosen -
  • the host cell may or may not be genetically modified to cause over-expression of four- of the above ER luminal localised chaperones.
  • the host cell may or may not be genetically modified to cause over-expression of four- of the above ER luminal localised chaperones.
  • one of the following combinations may or may not be chosen -
  • the host cell may or may not be genetically modified to cause over-expression of five of the above ER luminal localised chaperones.
  • the host cell may or may not be genetically modified to cause over-expression of five of the above ER luminal localised chaperones.
  • one of the following combinations may or may not be chosen -
  • the host cell may or may not be genetically modified to - cause over-expression of all six of the above.
  • ER luminal localised chaperones In other words, the following combination may or may not be chosen -
  • the host cell may or may not be genetically modified to cause over-expression of two, three or four helper proteins selected from LHSl, SILl 5 JEMl and SCJl 5 such as one of the following combinations -
  • LHSl and SILl LHSl and SILl; LHSl and JEMl; LHSl and SCJl; SILl and JEMl; SILl and SCJl; JEMl and SCJl; LHSl, SILl and JEMl; LHSl 5 SILl and SCJl; LHSl 5 JEMl and SCJl; SILl 5 JEMl and SCJl; or LHSl 5 SILl 5 JEMl and SCJl.
  • Chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation include SSAl 5 SSA2, SSA3, SSA4, SSEl 5 SSE2, SSBl 5 SSB2. A detailed description of these proteins and their genes is given separately below.
  • rtie host cell may or may not be genetically modified to cause over-expression of one of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • SSEl may or may not be chosen.
  • the host cell may or may not be genetically modified to cause over-expression of two of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • two of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be chosen -
  • SSAl in combination with one of SSA2, SSA3, SSA4, SSEl 5 SSE2, SSBl 5 SSB2; SSA2 in combination with one of SSA3, SSA4, SSEl, SSE2, SSBl 5 SSB2; SSA3 in combination with one of SSA4 5 SSEl 5 SSE2, SSBl 5 SSB2; ⁇ SSA4 in combination with one of SSEl 5 SSE2, SSBl 5 SSB2; SSEl in combination with one of SSE2, SSBl 5 SSB2;
  • SSE2 in combination with one of SSBl, SSB2; or SSBl in combination with SSB2.
  • the host cell may or may not be genetically modified to cause over-expression of three of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • the following combinations may or may not be chosen -
  • the host cell may or may not be genetically modified to cause over-expression of four of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • one of the following combinations may or may not be chosen — SSAl, SSA2, SSA3 and SSA4; SSAl 5 SSA2, SSA3 and SSEl; SSAl, SSA2, SSA3 and SSE2; SSAl 5 SSA2, SSA3 and SSBl; SSAl, SSA2, SSA3 and SSB2; SSAl, SSA2 5 SSA4 and SSEl; SSAl 5 SSA2, SSA4 and SSE2; SSAl, SSA2, SSA4 and SSBl; SSAl, SSA2, SSA4 and SSB2; SSAl 5 SSA2, SSEl and SSE2; SSAl, SSA2, SSEl and SSE2; SSAl, SSA2, SSEl and SSE2; SSAl, SSA
  • SSA4 SSEl, SSE2 and SSBl; SSA4, SSEl, SSE2 and SSB2; SSA4, SSEl, SSBl and SSB2; SSA4, SSE2, SSBl and SSB2; orSSEl, SSE2, SSBl andSSB2.
  • the host cell may or may not be genetically modified to cause over-expression of five of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • five of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be chosen -
  • SSAl, SSA2, SSA3, SSA4 and SSEl SSAl, SSA2, SSA3, SSA4 and SSE2
  • SSAl, SSA2, SSA3, SSA4 and SSBl SSAl 5 SSA2, SSA3, SSA4 and SSB2;
  • SSAl 5 SSA2, SSA3, SSEl and SSB2; SSAl, SSA2, SSA3, SSE2 and SSBl;
  • SSAl SSA2, SSA3, SSE2 and SSB2
  • SSAl 5 SSA2, SSA3, SSBl and SSB2
  • SSAl SSA2, SSEl 5 SSBl and SSB2; SSAl, SSA2, SSE2, SSBl and SSB2;
  • SSAl SSA3, SSA4, SSE2 and SSB2
  • SSAl 5 SSA3, SSA4, SSBl and SSB2;
  • the host cell may or may not be genetically modified to cause over-expression of six of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • six of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be chosen -
  • the host cell may or may not be genetically modified to cause over-expression of seven of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • seven of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be chosen —
  • the host cell may or may not be genetically modified to cause over-expression of all eight of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation.
  • the following combination may or may not be chosen -.
  • SSAl SSA2, SSA3, SSA4, SSEl, SSE2, SSBl and SSB2.
  • Mitochondrial chaperone and translocation proteins include ECMlO 5 MDJl, MDJ2. A detailed description of these proteins and their genes is given separately below.
  • the host cell may or may not be genetically modified to cause over-expression of one of the above mitochondrial chaperone and translocation proteins.
  • the host cell may or may not be genetically modified to cause over-expression of two of the above mitochondrial chaperone and translocation proteins.
  • the host cell may or may not be genetically modified to cause over-expression of two of the above mitochondrial chaperone and translocation proteins.
  • one of the following combinations may or may not be chosen -
  • ECMlO and MDJl ECMlO and MDJ2; ECMlO and MDJ2; or MDJl and MDJ2.
  • the host cell may or may not be genetically modified to cause over-expression of all three of the above mitochondrial chaperone and translocation proteins. In that case the following combination may or may not be chosen — 02289
  • the host cell may or may not be genetically modified to cause simultaneous over-expression of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, " fourteen, fifteen, sixteen or seventeen of the chaperones selected from the group consisting of JEMl, LHSl, SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl and MDJ2.
  • the chaperones selected from the group consisting of JEMl, LHSl, SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl and MDJ2.
  • the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined chaperones
  • it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins and the one or two other helper proteins may or may not be helper proteins involved in disulphide bond formation or protein degradation, as discussed below.
  • Over-expression of one (or more) of the ER luminal localised chaperones may or may not be combined with the over-expression of at least one of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation and/or the over-expression of at least one of the mitochondrial chaperone and translocation proteins.
  • any one of the following combinations may or may not be chosen -
  • SCJl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
  • KAR2 in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SS A3, SS A4, SSEl 5 SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
  • SILl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl 5 SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO 5 MDJl and MDJ2; or
  • one (or more) of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be simultaneously over-expressed with at least one of the ER luminal localised chaperones and/or at least one of the mitochondrial chaperone and translocation proteins.
  • the following combinations may or may not be chosen -
  • SSA2 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMlO, MDJl and MDJ2;
  • one of the mitochondrial chaperone and translocation proteins may or may not be simultaneously over-expressed with at least one of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation and/or at least one of the ER luminal localised chaperones.
  • one of the following combinations may or may not be chosen —
  • MDJ2 in combination with all six of JEMl , LHS 1, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2.
  • representative members of each of the above three groups of chaperone proteins may or may nqt be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • JEMl, SSAl and ERMlO JEMl, SSAl and MDJl
  • JEMl, SSAl and MDJ2 JEMl, SSA2 and ERMlO
  • JEMl, SSA2 and MDJl JEMl, SSA2 and MDJ2;
  • JEMl, SSA3 and ERMlO JEMl, SSA3 and MDJl
  • JEMl, SSA3 and MDJ2 JEMl, SSA3 and MDJ2;
  • JEMl, SSA4 and ERMlO JEMl, SS A4 and MDJl
  • JEMl, SSA4 and MDJ2 JEMl, SSA4 and MDJ2;
  • JEMl, SSEl and ERMlO JEMl, SSEl and MDJl
  • JEMl, SSEl and MDJ2 JEMl, SSEl and MDJ2;
  • JEMl, SSE2 and ERMlO JEMl, SSE2 and MDJl
  • JEMl, SSE2 and MDJ2 JEMl, SSBl and ERMlO
  • JEMl, SSBl and MDJl JEMl, SSBl and MDJ2;
  • JEMl, SSB2 and ERMlO JEMl, SSB2 and MDJl
  • JEMl, SSB2 and MDJ2 JEMl, SSB2 and MDJ2;
  • LHSl, SSA2 and ERMlO LHSl, SSA2 and MDJl
  • LHSl, SSA2 and- MDJ2 LHSl, SSA2 and- MDJ2
  • LHSl, SSA3 and ERMlO LHSl, SSA3 and MDJl LHSl, SSA3 and. MDJ2; LHSl, SSA4 and ERMlO; LHSl, SSA4 and MDJl; LHSl, SSA4 and;MDJ2;
  • LHSl, SSEl and ERMlO LHSl, SSEl and MDJl
  • LHSl, SSEl and MDJ2 LHSl, SSEl and MDJ2;
  • SCJl 5 SSAl and ERMlO SCJl 5 SSAl and MDJl
  • SCJl 5 SSAl and MDJ2 SCJl 5 SSAl and MDJ2;
  • SCJl 5 SSA2 and ERMlO SCJl 5 SSA2 and MDJl
  • SCJl, SSA2 and MDJ2 SCJl, SSA3 and ERMlO
  • SCJl, SSA3 and MDJl SCJl, SSA3 and MDJ2;
  • SCJl 5 SSE2 and ERMlO SCJl 5 SSE2 and MDJl; SCJl 5 SSE2 and MDJ2;
  • KAR2, SSE2 and ERMlO KAR2, SSE2 and MDJl
  • KAR2, SSE2 and MDJ2 KAR2, SSE2 and MDJ2;
  • helper proteins in particular helper proteins involved in disulphide bond formation or helper proteins involved in protein degradation, as discussed below.
  • Proteins involved hi the formation of disulphide bonds in other proteins include EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl. A detailed description of these proteins and their genes is given separately below.
  • one of the above disulphide bond formation proteins may or may not be over-expressed in the host cell.
  • ERV2 may or may not be chosen.
  • two of the above disulphide bond formation proteins may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • helper proteins may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen —
  • EROl, ERV2 and EUGl EROl, ERV2 and MPDl
  • EROl, ERV2 and MPD2 EROl, ERV2 and MPD2;
  • EROl, ERV2 and EPSl EROl, ERV2 and PDIl
  • EROl, EUGl and MPDl EROl, EUGl and MPDl
  • ERV2, EUGl and PDIl ERV2, MPDl and MPD2
  • ERV2, MPDl and EPSl ERV2, EUGl and PDIl
  • ERV2, MPDl and MPD2 ERV2, MPDl and EPSl
  • ERV2, MPDl and PDIl ERV2, MPD2 and EPSl
  • ERV2, MPD2 and PDIl ERV2, MPD2 and PDIl
  • MPDl 5 EPSl and PDIl; or MPD2, EPSl and PDIl.
  • helper proteins may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • MPD2 and EPSl ERV2, MPDl 5 MPD2 and PDIl; ERV2, MPDl 5 EPSl and PDIl; ERV2, MPD2, EPSl and PDIl; EUGl, MPDl 5 MPD2 and EPSl; EUGl 5 MPDl 5 MPD2 and PDIl; EUGl 5 MPDl 5 EPSl and PDIl; EUGl 5 MPD2, EPSl and PDIl; or MPDl 5 MPD2, EPSl and PDIl.
  • five of the above helper proteins may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • ERV2, EUGl, MPDl 5 MPD2 and PDIl ERV2 5 EUGl 5 MPDl, EPSl and PDIl;
  • helper proteins may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • EROl and ERV2 may function independently of each other or they may co-operate. Therefore, in one embodiment disclosure of EROl may or may not also include the combinations of EROl and ERV2, or ERV2 in its place. Similarly, in another embodiment disclosure of ERV2 may or may not also include the combinations of ERV2 and EROl, or EROl in its place.
  • all seven of the above helper proteins may or may not be simultaneously over-expressed in the host cell. In that case, the following combinations may or may not be chosen -
  • EROl EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
  • the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined disulphide bond formation helper proteins
  • it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins and the one or two other helper proteins may or may not be chaperones or helper proteins involved in protein degradation, as discussed above, and below, respectively.
  • helper proteins is a protein disulphide isomerase, such as a yeast and mammalian PDI, mammalian Erp59, mammalian prolyl-4-hydroxylase B- subunit, yeast GSBP, yeast EUGl and mammalian T3BP, then it may or may not be preferred, in one embodiment, to avoid co-expression with KAR2 or an equivalent thereof including hsp chaperone proteins such as other yeast Hsp70 proteins.
  • BiP, SSAl -4 a protein disulphide isomerase
  • SSBL SSCl and SSDl gene products and eukaryotic hsp70 proteins such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP) 3 glucose-regulated proteins 75, 78 and 80 (GRP75, GPR78 and GRP80) and the like, particularly where these are the sole helper proteins that are overexpressed in the host cell.
  • hsp70 proteins such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP) 3 glucose-regulated proteins 75, 78 and 80 (GRP75, GPR78 and GRP80) and the like, particularly where these are the sole helper proteins that are overexpressed in the host cell.
  • Proteins involved in protein degradation include DERI, DER3, HRD3, UJBC7 and D0A4. A detailed description of these proteins and their genes is given separately below.
  • one of the above proteins involved in protein degradation may or may not be over-expressed in the host cell.
  • DERI may or may not be chosen
  • DER3 may or may not be chosen
  • HRD3 may or may not be chosen
  • UBC7 may or may not be chosen
  • D0A4 may or may not be chosen.
  • two of the above proteins involved m protein degradation may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • DERI and DER3 DERI and HRD3; DERI and UBC7; DERI and D0A4; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; HRD3 and UBC7; HRD3 and D0A4; or UBC7 and D0A4.
  • three of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • four of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell.
  • one of the following combinations may or may not be chosen -
  • all five of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell.
  • the following combination is chosen - DERl 5 DER3, HRD3, UBC7 andDOA4.
  • the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined protein degradation helper proteins
  • it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins in total and the one or two other helper proteins may or may not be chaperones or disulphide bond formation helper proteins, as discussed above.
  • HACl encoded by a spliced or unspliced polynucleotide
  • HACl unfolded-protein response pathway regulator
  • HACl Over-expression of HACl can be achieved, for example, by the introduction of a recombinant polynucleotide that comprises the endogenous HACl gene coding sequence or a truncated intronless HACl coding sequence (Valkonen et al. 2003, Applied Environ. Micro., 69, 2065). A detailed description of this protein and its gene is given separately below. The same techniques can be used to over-express PTC2 or IREl.
  • a host cell of the present invention may or may not be genetically engineered to cause over-expression HACl, PTC2 or IREl , such as by modification of an endogenous gene encoding HACl, PTC2 or IREl, or by transformation with a recombinant gene encoding HACl 5 PTC2 or IREl.
  • HACl, PTC2 or IREl may or may not be simultaneously over-expressed with any of the above-defined combinations of other helper proteins.
  • the host cell of the present invention is not genetically engineered to cause HACl over-expression, such as by modification of an endogenous HACl gene or transformation with a recombinant HACl gene.
  • the host cell is additionally genetically modified by the introduction of at least one recombinant gene encoding at least one other helper protein, such as a DnaJ-like protein, an Hsp70 family protein and/or SILl or by the modification of the sequence of an endogenous gene encoding one or more other helper proteins at least one of a DnaJ-like protein, an Hsp70 family protein (such as LHSl) and SILl to cause increased expression of the thus modified gene.
  • helper protein such as a DnaJ-like protein, an Hsp70 family protein and/or SILl
  • the present invention also encompasses simultaneous over-expression of any combination of helper proteins derived from any of the above-defined groups.
  • helper proteins may or may not be simultaneously over- expressed.
  • Suitable combinations include any one of the following combinations:
  • JEMl and LHSl JEMl and SCJl
  • JEMl and KAR2 JEMl and SILl
  • JEMl and SILl JEMl and
  • JEMl and SSEl JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and
  • LHSl and SILl LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl
  • SSAl and DER3 SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA
  • the present invention encompasses simultaneous over-expression of at least three helper proteins, and that the at least three helper proteins may or may not be taken from any combination of helper proteins derived from any of the above-defined groups.
  • the at least three helper proteins may or may not be simultaneously over-expressed, with or without the over-expression of one or more additional helper proteins:
  • JEMl in combination with any one of the following combinations: LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and
  • SILl and ERV2 SILl and EUGl
  • SILl and MPDl SILl and MPD2
  • SILl and MPD2 SILl and MPD2
  • EPSl ⁇ EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and D0A4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and
  • SSA4 and ERV2 ⁇ SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; .
  • LHSl in combination with any one of the following combinations: JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; SCJl and KAR2; SCJl and
  • KAR2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and
  • SSA2 and DER3 SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SS A3 and EPSl; SS A3 and PDIl; SS A3 and DERI; SS A3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and E
  • SILl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and FKB2;- JEMl and SSAl; JEMl and SSA2; JEMl and SS A3; JEMl and SS A4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHS
  • SSA3 and EUGl SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; S5A4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SS A4 and EROl; SSA4 and ERV2; SS A4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl;
  • JEMl and SSA2 JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and
  • ERV2 and PDIl ERV2 and DERI
  • ERV2 and DER3 ERV2 and HRD3
  • ERV2 and UBC7 ER.V2 and DOA4
  • ERV2 and HACl EUGl and MPDl
  • EUGl and MPDl EUGl and
  • EUGl and HRD3 EUGl and UBC7; EUGl and DOA4; EUGl and HACl;
  • MPDl and MPD2 MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and
  • MPD2 and HRD3 MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
  • HACl HACl ; or D0A4 and HACl .
  • SSAl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
  • JEMl and SSA2 JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and
  • JEMl and SSBl JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and ,
  • LHSl and SSA2 FKB2; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and
  • SCJl and MDJ2 SCJl and EROl
  • SCJl and ERV2 SCJl and EUGl
  • SCJl and EROl SCJl and EROl
  • SCJl and ERV2 SCJl and EUGl
  • SCJl and EUGl SCJl and EUGl
  • MDJl and MPDl 20 MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; .
  • MD J2 and EPSl MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and
  • ERV2 and EUGl ERV2 and MPDl
  • ERV2 and MPD2 ERV2 and MPD2
  • ERV2 and EPSl ERV2 and PDIl
  • ERV2 and DERI ERV2 and DER3
  • ERV2 and HRD3 ERV2
  • SSA2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and ' SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl
  • KAR2 and SILl KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE
  • EPSl and PDIl EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
  • SSA3 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • SCJl and SSAl SCJl and SSA2; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl;
  • SSA4 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • SILl and DER3 SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FICB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SS A3; SSAl and SSEl;
  • JEMl and LHSl JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR
  • SSE2 and SSBl SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and
  • SSBl and SSB2 SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and
  • SSBl and HRD3 SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and -EROl; SSB2 and
  • ERV2 ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl;
  • SSBl and DERI SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl;
  • JEMl and LHSl JEMl and SCJl
  • JEMl and KAR2 JEMl and SILl
  • JEMl and FKB2 JEMl and SSAl
  • JEMl and SSA2 JEMl and SSA3
  • JEMl and SSA4 JEMl and .
  • JEMl and SSE2 JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4;
  • SSAl and DOA4 SSAl and HACl
  • SSA2 and SSA3 SSA2 and SSA4
  • SSA2 and SSEl SSA2 and SSE2
  • SSA2 and SSB2 SSA2 and ECMlO
  • SSA2 and DOA4 SSAl and HACl
  • SSA2 and HACl SSA2 and HACl
  • SSA2 and SSA3 SSA2 and SSA4
  • SSA2 and SSEl SSA2 and SSE2
  • SSA2 and SSB2 SSA2 and ECMlO
  • SSA2 and DOA4 SSAl and HACl
  • SSA2 and SSA3 SSA2 and SSA4
  • SSA2 and SSEl SSA2 and SSE2
  • SSA2 and SSB2 SSA2 and ECMlO
  • SSA2 and DOA4 SSAl and HACl
  • SSA2 and SSA3 SSA2 and SSA4
  • SSA2 and SSEl
  • MDJl MDJl
  • SSA2 and MDJ2 SSA2 and EROl
  • SSA2 and ERV2 SSA2 and EUGl
  • SSA2 and MPDl SSA2 and MPD2
  • SSA2 and MPD2 SSA2 and MPD2
  • SSA2 and EPSl SSA2 and PDIl
  • SSA2 and MPDl SSA2 and MPD2
  • SSA3 and ERV2 SSA3 and EUGl
  • S S A3 and MPDl S S A3 and MPDl
  • SSA3 and MPD2 S S A3 and MPDl
  • SSA3 and UBC7 SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl;
  • JEMl and LHSl JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • ECMlO in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJLl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and
  • LHSl and EUGl LHSl and MPDl
  • LHSl and MPD2 LHSl and MPD2
  • LHSl and EPSl LHSl and PDIl
  • LHSl and DERI LHSl and DER3
  • LHSl and HRD3 LHSl and UBC7
  • LHSl and D0A4 LHSl and HACl
  • SCJl and KAR2 SCJl and SILl
  • MDJ2 and EROl . MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPD 1 ; ERV2 and MPD2; ERV2 and EPS 1 ; ERV2 and PDIl ;
  • MDJl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • KAR2 and SILl KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SS A3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl ; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl;
  • SSA2 and HACl SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJ2; SSA3 and EROl;
  • SSA3 and ERV2 SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4;.
  • SSA4 andMPD2 SSA4 and EPSl
  • SSA4 and PDIl SSA4 and DERI
  • SSA4 and MPD2 SSA4 and EPSl
  • SSA4 and PDIl SSA4 and PDIl
  • SSA4 and DERI SSA4 and MPD2
  • MDJ2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • SSA2 and HACl SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and EROl;
  • SSA3 and ERV2 SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSA
  • SSE2 and SSBl SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and
  • SSE2 and EPSl SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2;
  • SSBl and DOA4 SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and
  • MDJl and DERI MDJl and DERI
  • MDJl and DER3 MDJl and HRD3; KdDJl and UBC7
  • MDJl and DOA4 MDJl and HACl
  • EROl and ERV2 EROl and EUGl
  • EROl and MPDl EROl and MPD2
  • EROl and EPSl EROl and PDIl
  • EROl and DERI EROl and DERI;
  • EROl and DER3 EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and
  • ERV2 and UBC7 ERV2 and DOA4
  • ERV2 and HACl EUGl and MPDl
  • EUGl and MPD2 EUGl and MPD2
  • EUGl and EPSl EUGl and PDIl
  • EUGl and DERI EUGl and
  • MPDl and DER3 MPDl and HRD3; MPDl and UBC7; MPDl and DOA4;
  • MPDl and HACl MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and.
  • EPSl and UBC7 EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and
  • EROl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and K
  • SSE2 and EPSl SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and D0A4; SSE2 and HACl; SSBl and SSB2;
  • SSBl and ECMlO SSBl and MDJl; SSBl and MDJ2; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and
  • PDIl PDIl
  • SSBl and DERI SSBl and DER3
  • SSBl and HRD3 SSBl and UBC7
  • SSBl and DOA4 SSBl and HACl
  • SSB2 and ECMlO SSB2 and MDJl
  • SSB2 and MDJ2 SSB2 and MDJ2
  • SSB2 and ERV2 SSB2 and EUGl
  • MDJl and DERI MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and
  • MPDl MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI;
  • MDJ2 and DER3 MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2. and
  • EPSl ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl ; EUGl and MPDl ; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and
  • MPDl and DER3 MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and
  • EPSl and UBC7 EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and
  • ERV2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJXl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DO
  • SIL1 and SSA4 SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and EUGl; SHl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SJLl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and
  • SSA4 FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and EU
  • EUGl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
  • JEMl and SSAl JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl
  • ECMlO and MPD2 ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI;
  • ECMlO and DER3 ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4;
  • ECMlO and HACl ECMlO and HACl
  • MDJl and MDJ2 MDJl and EROl
  • MDJl and ERV2 MDJl and MPDl
  • MDJl and MPD2 MDJl and MPD2
  • MDJl and EPSl MDJl and PDIl
  • MDJl and DERI MDJl and DER3
  • MDJl and HRD3 MDJl and UBC7; MDJl and
  • EROl and PDIl EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and MPDl; ERV2 and
  • ERV2 and HRD3 ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and
  • MPD2 and HRD3 MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
  • MPDl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJXl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl;.
  • MPD2 in combination with any one of "the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMI and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4; JEMl and HACl ; LHS 1 and SCJl ; LHS
  • MDJ2 and PDIl MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUV2 and EUGl; ERV2 and MPDl;
  • EPSl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
  • JEMl and SSAl JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and
  • LHSl and SSEl LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and
  • SCJl and SSAl SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2
  • JEMl and LHSl JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR
  • ERV2 and EUGl ERV2 and MPDl
  • ERV2 and MPD2 ERV2 and MPD2
  • ERV2 and EPSl ERV2 and DERI
  • ERV2 and DER3 ERV2 and HRD3
  • ERV2 and UBC7 HACl
  • ERV2 and DOA4 ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and DERI; EPSl and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
  • JEMl and LHSl JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; JEMl and SILl; JEMl and
  • KAR2 and SILl KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; ICAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; K
  • MDJl and MPD2 MDJl and EPSl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and
  • MDJ2 and EPSl MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 5 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; 0 ERV2 and DOA4; ERV2 and HACl ; EUGl and MPDl ; EUGl and MPD2; EUG
  • JEMl and LHSl JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR
  • HACl HACl; EPSl and PDIl; EPSl and DERI; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl .
  • HRD3 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; -JEM1 and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl
  • SSE2 and EUGl SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3;; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2
  • SSA4 FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FICB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl
  • MDJl and MPD2 10 MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and
  • D0A4 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
  • JEMl and SSAl JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and HACl ; LHS 1 and SCJl ; LHS 1 and KAR2; LHS 1 and SILl ; LHS 1 and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4;
  • FKB2 and ERV2 FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3;
  • HACl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SIXl; JEMl and FKB2; JEMl and SSAl; JEMl and SS A2; JEMl and SS A3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and
  • any protein can be expressed as the protein product of choice.
  • the protein product of choice may or may not be a protein that is naturally produced by the host cell, in which case the protein may or may not be encoded by the host cell's endogenous gene for that protein or the protein may or may not be encoded (fully, or in part) by an exogenous polynucleotide sequence.
  • a recombinant or genetically modified endogenous gene has a sequence that is different to the endogenous genetic material of the host cell.
  • the protein product of choice may or may not be a heterologous protein, by which we mean that the protein is one that is not naturally produced by the host cell.
  • the protein may or may not be encoded by an exogenous polynucleotide sequence.
  • the protein product of choice 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 that encodes the protein product of choice.
  • the protein product of choice may or may not be intracellular.
  • WO 2005/061718 (Example 12) describes the co-over- expression of the cytoplasmic chaperone SSAl and a secreted recombinant transferrin, in order to increase the production of the secreted .recombinant transferrin.
  • the protein product of choice comprises the sequence of a eukaryotic protein, or a fragment or variant thereof.
  • Suitable eukaryotes include fungi, plants and animals.
  • the protein product of choice is a fungal protein, such as a yeast protein.
  • the protein product of choice is an animal protein.
  • Exemplary animals include vertebrates and invertebrates.
  • Exemplary vertebrates include mammals, such as humans, and non-human mammals.
  • the protein product of choice may or may not comprise the sequence of a yeast protein.
  • the protein product of choice may or may not comprise 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 a 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 aprotinin, amyloid precursor protein and those described hi WO 03/066824, with or without albumin fusions
  • interferons such as interferon ⁇ species and sub-species, interferon ⁇ species and sub-species, interferon ⁇ species and sub-species, interferon ⁇ species and sub-species
  • interleukins such as ILlO, ILIl and IL2
  • leptin CNTF and fragment thereof (such as CNTF A ⁇ i 5 ⁇ AxokineTM)
  • ILl-receptor antagonist such as erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin
  • 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 is intended combinations such as VaI 3 He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy 5 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 firom 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 a!., (1994) Nucleic Acids Res., 22(22), 4673-80).
  • the parameters used may be as follows:
  • the fragment may comprise at most 5, 10, 20, 30, 40 or 50% of the complete sequence of the full mature polypeptide.
  • 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.
  • the protein product of choice 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
  • 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.
  • the term “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.
  • 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
  • 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.
  • Swissprot accession number P35747 rhesus monkey (e.g. see Swissprot accession number Q28522), mouse (e.g. see Swissprot accession number 089020), 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.
  • albumin Many naturally occurring mutant forms of albumin are known. Many are described in Peters, (1996, AU 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-stabiUty) has not significantly been ' changed.
  • 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-range
  • 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 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.
  • 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 et al., 1994). The parameters used may 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.
  • 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), osmolality (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
  • osmolality oncotic pressure, colloid osmotic pressure
  • behaviour in a certain pH-range pH-range
  • a fragment will 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
  • domain I was defined as consisting of amino acids 1-197
  • domain II was defined as consisting of amino acids 189-385
  • domain III was defined as consisting of amino acids 381-585. Partial overlap of the domains occurs because of the extended ⁇ -helix structure (hl ⁇ -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, IIIA and IIIB).
  • Sub- domain IA comprises amino acids 6-105
  • sub-domain IB comprises amino acids 120-177
  • sub-domain ILA comprises amino acids 200-291
  • sub-domain HB comprises amino acids 316-369
  • sub-domain IIIA comprises amino acids 392-491
  • sub-domain IIIB 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 protein product of choice 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 5 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.
  • 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. 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.
  • 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 immunoglobulins or a variant or fragment of any of these may 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-terrninal 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 U S A, 92, 2820, AIi, et al., 1999, J Biol Chem, 274, 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 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 be presented alone or as fusion partners for albumin, the Fc region of immunoglobulin, transferrin, lactoferrin or any other protein or fragment or variant of any of the above, as a desired polypeptide.
  • the protein product of choice may or may not be a therapeutically active protein.
  • the protein product of choice may or may not comprise a leader sequence effective to cause secretion in the host cell (such as in a yeast host 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 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. (1985) Science, 229, 1219-1224) and heat-shock protein-150 (Hspl50p) (see WO 95/33833). Additionally, leader sequences from the S.
  • MFa- 1 cerevisiae mating factor alpha-1 protein
  • HSA human serum albumin
  • MFa- 1 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.
  • Modified leader sequences are also disclosed in the examples of this application and 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 protein products of choice.
  • the protein product of choice comprises disulphide bonds in its mature form.
  • the disulphide bonds may be intramolecular and/or mtermolecular.
  • the protein product of choice may or may not be a commercially useful protein.
  • Some 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 as a protein, but that that effect is not the main or sole reason for expressing the protein therein.
  • the host cell may be any type of cell.
  • the host cell may or may not be an animal (such as mammalian, avian, insect, etc.), plant, fungal or bacterial cell.
  • Bacterial and fungal, such as yeast, host cells may or may not be preferred.
  • the host cell may or may not be an animal (such as mammalian, avian, insect, etc.) cell.
  • Suitable methods for transformation of animal cells are well known in the art and include, for example the use of retrovirus vectors (such as lentivirus vectors).
  • retrovirus vectors such as lentivirus vectors.
  • the host cell is a yeast cell, such as a member of the Saccharomyces, Kluyveromyces, or Pichia genus, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris and Pichia membranaefaciens, or Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Hansenula polymorpha (also known as Pichia angusta) or Kluyveromyces drosophilarum are preferred.
  • Saccharomyces, Kluyveromyces, or Pichia genus such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris and Pichia membranaefaciens, or Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosacchar
  • 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 T/GB2006/002289 reported to be modified, in nature, by O-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 hi 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.
  • the yeast could be transformed to produce an anti-Pmt agent, such as an anti-Pmt antibody.
  • the yeast could be cultured in the presence of a compound that inhibits the activity of one of the PMT genes (Duffy et al, "Inhibition of protein mannosyltransf erase 1 (PMTl) activity in the pathogenic yeast Candida albicans", International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, 26-31 August 2001, Monte Verita, Switzerland, Poster Abstract P38; the poster abstract may be viewed at http://www.micro.biol.ethz.ch/cellwall/).
  • PMTl protein mannosyltransf erase 1
  • disruption of one or more of the genes equivalent to the PMT genes of S. cerevisiae is also beneficial, e.g. hi Pichia pastoris or Kluyverojnyces lactis.
  • the sequence of PMTl (or any other PMT gene) isolated from S. cerevisiae may 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 also have a deletion of the HSP 150 and/or YAP 3 genes as taught respectively in WO 95/33833 and WO 95/23857.
  • the host cell type may be selected for compatibility with the plasmid type being used.
  • any suitable plasmid may be used, such as a centromeric plasmid.
  • the examples provide suitable plasmids (centromeric YCplac33-based vectors) for use to transform yeast host cells of the present invention.
  • any other suitable plasmid may be used, such as a yeast- compatible 2 ⁇ m-based plasmid.
  • Plasmids obtained from one yeast type can be maintained in other yeast types (Irie et al, 1991, Gene, 108(1), 139-144; Irie et al, 1991, MoI. Gen. Genet, 225(2), 257-265).
  • pSRl from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae.
  • the plasmid may or may not be a 2 ⁇ m-family plasmid and the host cell will be compatible with the 2 ⁇ m-family plasmid used (see below 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 pELDl 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 Saccharomyces cerevisiae or Saccharomyces carlsbergensis.
  • the plasmid may be based on the 2 ⁇ m plasmid and the yeast cell may be Saccharomyces cerevisiae.
  • a 2 ⁇ m ⁇ famiry plasmid can be said to be "based on" a naturally occurring plasmid if it comprises one, two or preferably three of the genes FiP, REPl and REP2 having sequences derived from that naturally occurring plasmid.
  • a plasmid as defined above may be introduced into a host through standard techniques.
  • transformation of prokaryotic host cells see, for example, Cohen et al (1972) Proc. Natl Acad. ScI 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 Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. Methods for the transformation of S.
  • Electroporation is also useful for transforming cells and is well known in the art for transforming fungal (including yeast) cell, plant cells, bacterial cells and animal (including vertebrate) cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol 194, 182.
  • a plasmid will transform not all of the hosts and it will therefore be necessary to select for transformed host cells.
  • a plasmid may 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.
  • Typical yeast selectable marker include LEU2, TRPl, HIS3, HIS4, URA3, URA5, SFAl, ADE2, METIS, LYS5, LYS2, ILV2, FBAl, PSEl, PDU and PGKl.
  • any gene whose chromosomal deletion or inactivation results in an unviable 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, 119).
  • 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 additions or modifications to the growth medium.
  • essential marker genes in the context of the present application are those that, when deleted or inactivated hi a host cell, result in a deficiency which cannot be complemented by additions or modifications to the growth medium.
  • a plasmid may 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, neomycin or zeocin resistance for eukaryotic cell culture, and tetracycline, kanamycin, ampicillin (i.e. ⁇ -lactamase) or zeocin resistance genes for culturing in E. coli and other bacteria.
  • Zeocin resistance vectors are available from Invitrogen.
  • 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 a plasmid, optionally to allow the expression of a recombinant polypeptide (i.e. a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is heterologous to the host cell, in the sense that that polypeptide is not naturally produced by the host).
  • a recombinant polypeptide i.e. a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is heterologous to the host cell, in the sense that that polypeptide 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 MoL Biol. 98, 503 or Berent et al (1985) Biotech. 3 5 208 or other methods of DNA and RNA analysis common in the art.
  • successful transformation can be confirmed by well known immunological methods when the recombinant DNA is capable of directing the expression of the protein.
  • 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.
  • transformed host cells preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.
  • transformed cells may 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 can be used as capsules for a huge variety of applications, such as fragrances, flavours and pharmaceuticals.
  • Transformed host cells may 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 the helper protein(s) and the protein product of choice.
  • the culture medium may be non-selective or place a selective pressure on the maintenance of a plasmid.
  • the thus produced protein product of choice may be present intracellularly or, if secreted, in the culture medium and/or periplasmic space of the host cell.
  • the present invention also provides a method for producing a protein product of choice, the method comprising: (a) providing a host cell of the invention comprising a polynucleotide encoding protein product of choice as defined above; and
  • the step of growing the host cell may or may not involve allowing a host cell derived from a multicellular organism to be regrown into a multicellular recombinant organism (such as a plant or animal) and, optionally, producing one or more generations of progeny therefrom.
  • the method may or may not further comprise the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.
  • the step of "purifying the -thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium” optionally comprises cell immobilisation, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilisation, separation and/or breakage.
  • Cell immobilisation 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.
  • 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, hydroxyapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography (“HPLC”), reverse phase HPLC 5 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 purifying 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 lO ⁇ g.L 4 , lO "3 g.L 4 5 0.01 gl/ 1 , 0.02 gX 4 , 0.03 g.L 4 , 0.04 g.L 4 , 0.05 g.L 4 , 0.06 gX 4 ,0.07 g.L 4 , 0.08 g.L 4 , 0.09 g.L 4 , 0.1 g.L 4 , 0.2 g.L 4 , 0.3 g.L 4 , 0.4 gX 4 , 0.5 gX 4 , 0.6 g.L 4 , 0.7 gl ⁇ 0.8 .
  • gX 4 0.9 g.L "1 , 1 g.L 4 , 2 gX 4 , 3 g.L 4 , 4 gX 4 , 5 gX "1 , 6 g.L 4 , 7 g.L 4 , 8 9 g.L 4 , 10 g.L 4 , 15 g.L 4 , 20 g.L 4 , 25 gX 4 , 30 gX 4 , 4.0 g.L 4 ,50 g.L 4 , 60 g.L 4 , 70 gX 4 , 70 g.L 4 , 90 g.L 4 , 100 g.L 4 , 150 g.L 4 , 200 g.L 4 , 250 g.L 4 , 300 g.L 4 , 350 g.L 4 , 400 g.L 4 , 500 g.L 4 , 600 g.L 4 , 700 g.L 4 , 800 g.L 4 , 900
  • a commercially or industrially acceptable level of purity may be obtained by a relatively crude purification method by which the protein product of choice is put into a form suitable for its intended purpose.
  • a protein preparation that has been purified to a commercially or industrially acceptable level of purity may, in addition to the protein product of choice, also comprise, for example, cell culture components such as host cells or debris derived therefrom.
  • cell culture components such as host cells or debris derived therefrom.
  • high molecular weight components such as host cells or debris derived therefrom
  • the protein may or may not be 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 protein may be used for any of its known utilities, which, in the case of albumin, include i.v. administration to patients to treat severe burns, shock and blood loss, supplementing culture media, and as an excipient in formulations of other proteins.
  • a method of the present invention may or may not further comprise the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
  • a therapeutically useful protein obtained by a process of the invention is administered alone, it is preferable to present it as a pharmaceutical formulation, 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 not deleterious to the recipients 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 of the present invention may or may not further comprise the step of lyophilising the thus purified protein product of choice.
  • JEMl is one S. cerevisiae helper protein of interest for the present invention. It is also known as KAR8, and its gene is a non-essential gene located on chromosome X. It is a DnaJ-like chaperone and is thought to be required for nuclear membrane fusion during mating. It localises to the ER membrane and exhibits genetic interactions with Kar2p (described further below).
  • a published protein sequence for the protein Jemlp is as follows:
  • the ORF ofthe JEMl gene is 1.938 kbp in size.
  • a published nucleotide coding sequence ofJEMl is as follows, although it will be appreciated thatthe sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
  • JEMl we include fragments or variants thereof having equivalent JEMl-like activity.
  • Such variants may or may not include bacterial DnaJ proteins and/or may or may not include eukaryotic DnaJ type 2006/002289
  • JEMl proteins, such as other members of the Hsp40 family.
  • a variant of JEMl may not be SCJl.
  • LHSl is another S. cerevisiae helper protein of interest for the present invention. It is also known as CERl or SSIl, is encoded by a non-essential gene which is located on chromosome XI. It is thought to be a molecular chaperone of the endoplasmic reticulum lumen, involved in polypeptide translocation and folding. It is a member of the HSP70 family, localizes to the lumen of the ER, and is thought to be regulated by the unfolded protein response pathway.
  • the ORF of the LHSl gene is 2.646 kbp .in size.
  • a published nucleotide coding sequence of LHSl is as follows, 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:
  • LHSl LHSl
  • variants may or may not include bacterial DnaK proteins and/or eukaryotic DnaK type proteins, such as other members ofthe Hsp70 family.
  • SCJl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of bacterial chaperone DnaJ, located in the ER lumen where it cooperates with Kar2p (described below) to mediate maturation of proteins.
  • SCJl is encoded by a non-essential gene comprising an ORF of 1.134 kbp.
  • the gene is located on chromosome XIII.
  • a published nucleotide coding sequence of SCJl is as follows, 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:
  • SCJl we include fragments or variants thereof having equivalent SCJl -like activity.
  • KAR2 is another S. cerevisiae helper protein of interest for the present invention.
  • KAR2 is also known as BIP or GRP78.
  • Kar2p is an ATPase involved in protein import into the ER.
  • Kar2p also acts as a chaperone to mediate protein folding in the ER and may play a role in ER export of soluble proteins. It is also thought to regulate the unfolded protein response via interaction with Irelp.
  • a published protein sequence for the protein Kar2p is as follows: . •
  • KAR2 is encoded by an essential gene comprising an ORP that is 2.049 kbp in size and located on chromosome X.
  • ORP that is 2.049 kbp in size and located on chromosome X.
  • a published nucleotide coding sequence of KAR2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidentical proteinproduct:
  • KAR2 we include fragments or variants thereof having equivalent KAR2-like activity.
  • SILl is another S. cerevisiae helper protein of interest for the present invention and is also known as SLSl.
  • this helper protein was generally referred to as SLSl in UK patent application no. 0512707.1, from which this application claims priority; it will be understood by the person skilled in the art that reference in UK patent application no. 0512707.1 to SLSl and reference in this application to SILl should be taken to be reference to the same helper protein.
  • SILIp is an ER-localized protein required for protein translocation into the ER, which interacts with the ATPase domain of the Kar2p chaperone suggesting some role in modulating its activity. It is also thought to be a homolog of Yarrowia lipofytica SILl; and a GrpE-like protein in the ER.
  • a published protein sequence for the protein SILIp is as follows:
  • L* SILl is encoded by a non-essential gene comprising an ORP that is 1.226 kbp in size and is located on chromosome XV.
  • a published nucleotide coding sequence of 5ZLi is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
  • SILl we include fragments or variants (including homologues) thereof having equivalent SILl-like activity.
  • variants of SILl may or may not include bacterial GrpE type proteins and/or animal (such as mammalian) GrpE-like proteins.
  • Variants of SILl may be a nucleotide exchange factor for an Hsp70 family protein, which nucleotide exchange factor is optionally not an Hsp70 family protein in itself.
  • Suitable variants of SILl may or may not be FESl and/or MGEl.
  • a variant of SILl may or may not be localised to the lumen of the ER (such as SELl itself) to the mitochondria (such as MGEl) or to the cytosol (such as FESl).
  • a variant of SILl may or may not include proteins such as members so of the mammalian GrpE-like protein family, the NEF family or BAG-I (such as described in Hohfeld and Jentsch (1997) EMBO J. 16, 6209), mammalian BiP-associated protein (BAP) (Chung et al (2002) J Biol. Chem. 277, 47557), a human GrpE-like protein (e.g.
  • accession number AAG31605 the protein defined by accession number AAG31605 (Choglay et al (2001) Gene 267, 125), an ⁇ rabidopsis thaliana GrpE-like protein (for example, accession numbers AAK68792 and BAB08589) (Sato et al (1998) DNA Res. 5, 41), a Chlamydia trachomatis Protein grpE (HSP-70 cofactor) (e.g. accession number P36424), a Pongo pygmaeus adenine nucleotide exchange factor (e.g. accession number CAH89792), a Mus musculus mitochondrial GrpE-like 2 protein (e.g.
  • accession number NP_067271 a Mus musculus mitochondrial GrpE-like 1 protein (e.g. accession number NP_077798), a Gallus gallus GrpE protein homolog 2, mitochondrial precursor (Mt-GrpE#2) (e.g. accession number XP_425191), a Gallus gallus BiP-associated protein (e.g. accession number XP_414514), an Haemophilus influenzae 86-028NP GrpE protein (e.g. as defiend by accession number YP_247735) (Harrison et al (2005) J Bacteriol. 187, 4627), an Escherichia coli GrpE heat shock protein (e.g.
  • accession number NP_417104 (Riley et al (1997) Science 277, 1453), a Streptococcus pneumoniae GrpE heat shock protein (e.g. as defined by accession number AAD23453), a Bacillus subtilis GrpE protein accession number (e.g. as defined by BAA12463) (Mizuno et al (1996) Microbiology (Reading, Engl.) 142, 3103) and/or a Nicotiana tabacum chaperone GrpE type 1 or GrpE type 2 protein (e.g. as defined by accession numbers AAC72386 or AAC72387) (Padidam et al (1999) Plant MoI Biol. 39, 871).
  • Variants of SILl may have an activity equivalent to SILl, when co-expressed with one or both of JEMl and LHSl, for example in the manner as set out in the present examples.
  • a host cell of the present invention when genetically 89
  • modified to cause simultaneous over-expression of a variant of SILl with one or both of JEMl and LHSl will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl, the increase being compared to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl.
  • substantially the same increase in the production of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl (the increased being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
  • substantially the same reduction of fragmentation of a protein product we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
  • FKB2 is another S. cerevisiae helper protein of interest for the present invention and is also known as FPR2 and FKBPl 3.
  • Fkb2p is a membrane bound peptidyl- prolyl cis-trans isomerase (PPIase) that binds to the drugs FK506 and rapamycin.
  • PPIase membrane bound peptidyl- prolyl cis-trans isomerase
  • Fkb2p The expression pattern of Fkb2p suggests possible involvement in ER protein trafficking.
  • a published protein sequence for the protein Fkb2p is as follows:
  • FKB2 is encoded by a non-essential gene comprising an ORF that is 0.408 kbp in size and is located on chromosome IV.
  • a published nucleotide coding sequence of FKB2 is as follows, 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:
  • FKB2 we include fragments or variants thereof having equivalent FKB2-like activity.
  • SSAl is another S. cerevisiae helper protein of interest for the present invention and is also known as YGlOO.
  • Ssalp is an ATPase that is involved in protein folding and nuclear localization signal (NLS)-directed nuclear transport. It is a member of heat shock protein 70 (HSP70) family. It forms a chaperone complex with Ydjlp and is localized to the nucleus, cytoplasm, and cell wall
  • HSS70 heat shock protein 70
  • SSAl is encoded by a non-essential gene comprising an ORF that is 1.929 kbp in size and is located on chromosome I.
  • a published nucleotide coding sequence of SSAl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
  • SSAl we include fragments or variants thereof having equivalent SSAl -like activity.
  • SSA2 is another S. cerevisiae helper protein of interest for the present invention.
  • Ssa2p is an ATP binding protein that is involved in protein folding and vacuolar import of proteins; member of heat shock protein 70 (HSP70) family. It is associated with the chaperoriin-containing T-complex. It is present in the cytoplasm, vacuolar membrane and cell wall.
  • HSP70 heat shock protein 70
  • SSA2 is encoded by a non-essential gene comprising an ORF that is 1.920 kbp in size and is located on chromosome XII.
  • a published nucleotide coding sequence of SSA2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
  • SSA2 we include fragments or variants thereof having equivalent SSA2-like activity.
  • SS A3 is another S. cerevisiae helper protein of interest for the present invention, which is also known as HSP70.
  • Ssa3p is an ATPase involved in protein folding and the response to stress. It plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation and is a member of the heat shock protein 70 (HSP70) family.
  • S S A3 is localized to the cytoplasm.
  • a published protein sequence for the protein Ssa3p is as follows:
  • SSA3 is encoded by a non-essential gene comprising an ORF that is 1.950 kbp in size and is located on chromosome II.
  • a published nucleotide coding sequence of SSA3 is as follows, 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:
  • SSA4 is another S. cerevisiae helper protein of interest for the present invention.
  • Ssa4p is a heat shock protein that is highly induced upon stress. It plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation; member of the HSP70 family. It is a cytoplasmic protein that concentrates in nuclei upon starvation.
  • a published protein sequence for the protein Ssa4p is as follows:
  • SSA4 is encoded by anon-essential gene comprising an ORF that is 1.929 kbp in size and is located onchromosomeV.
  • Apublishednucleotide coding sequence of SSA4 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
  • SSA4 we include fragments or variants thereof having equivalent SSA4-like activity.
  • SSEl is another S. cerevisiae helper protein of interest for the present invention and is also known as LP G3 and MSI3.
  • S se Ip is an ATPase that is a component of the heat shock protein Hsp90 chaperone complex. It binds unfolded proteins and is a member of the heat shock protein 70 (HSP70) family. It is localized to the cytoplasm.
  • a published protein sequence for the protein Sselp is as follows:
  • SSEl is encoded by a non-essential gene comprising an ORF that is 2.082 kbp in size and is located on chromosome XVI.
  • a published nucleotide coding sequence of SSEl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identicalproteinproduct:
  • SSE2 is another S. cerevisiae helper protein of interest for the present invention.
  • Sse2p is a member of the heat shock protein 70 (HSP70) family. It may be involved in protein folding and is localised to the cytoplasm. It is highly homologous to the heat shock protein Sselp.
  • a published protein sequence for the protein Sse2p is as follows:
  • SSE2 is encoded by a non-essential gene comprising an ORP that is 2.082 kbp in size and is located on chromosome II.
  • a published nucleotide coding sequence of SSE2 is as follows, 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:
  • SSE2 we include fragments or variants thereof having equivalent SSE2-like activity.
  • SSBl is another S. cerevisiae helper protein of interest for the present invention and is also known as YGlOl.
  • Ssblp is a cytoplasmic ATPase that is a ribosome- associated molecular chaperone. It may be involved in the folding of newly- synthesized polypeptide chains and is a member of the heat shock protein 70 (HSP70) family. It interacts with the phosphatase subunit Reglp.
  • HSP70 heat shock protein 70
  • SSBl is encoded by a non-essential gene comprising an ORF that is 1.842 kbp in size and is located on chromosome IY.
  • a published nucleotide coding sequence of SSBl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
  • SSB2 is another S. cerevisiae helper protein of interest for the present invention.
  • Ssb2p is a cytoplasmic ATPase that is a ribosome-associated molecular chaperone. It may be involved in the folding of newly-synthesized polypeptide chains. It is a member of the heat shock protein 70 (HSP70) family and is a homolog of SSBl.
  • HSP70 heat shock protein 70
  • SSB2 is encoded by anon-essential gene comprising an ORF that is 1.842 kbp in size and is located on chromosome XTV.
  • Apublishednucleotide coding sequence of SSB2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
  • SSB2 we include fragments or variants thereof having equivalent SSB2-like activity.
  • ECMlO is another S. cerevisiae helper protein of interest for the present invention and is also known as SSC3.
  • EcmlOp is a heat shock protein of the Hsp70 family, which is localised in mitochondrial nucleoids. It is thought to play a role in protein translocation. It interacts with Mgelp in an ATP-dependent manner. Over-expression has been shown to induce extensive mitochondrial DNA aggregations.
  • ECMlO is encoded by a non-essential gene comprising an ORF that is 1.935 kbp in size and is located on chromosomeV.
  • Apublishednucleotide coding sequence ofECMlO is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
  • ECMlO we include fragments or variants thereof havingequivalentECMlO-like activity.
  • MDJl is another S. cerevisiae helper protein of interest for the present invention.
  • Mdj Ip is a protein involved in folding of mitochondrially synthesised proteins in the mitochondrial matrix. It localises to the mitochondrial inner membrane and is a member of the DnaJ family of molecular chaperones.
  • a published protein sequence for the protein Mdj Ip is as follows:
  • MD J2 is another S. cerevisiae helper protein of interest for the present invention.
  • Mdj2p is a protein of the mitochondrial inner membrane. Its function partially overlaps that of Mdjlp, which is a chaperone involved in folding of mitochondrially synthesised proteins in the mitochondrial matrix. It is a member of the DnaJ family.
  • a published protein sequence for the protein Mdj2p is as follows:
  • MDJ2 is encoded by a non-essential gene comprising an ORF that is 0.441 kbp in size and is located on chromosome XTV.
  • a published nucleotide coding sequence of MDJ2 is as follows, 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:
  • EROl is another S. cerevisiae helper protein of interest for the present invention.
  • Erolp is a glycoprotein required for oxidative protein folding in the endoplasmic reticulum.
  • a published protein sequence for the protein Erolp is as follows:
  • EROl is encodedby an essential gene comprising an ORP thatis 1.692 kbp in size and is located on chromosome XIII.
  • a published nucleotide coding sequence of EROl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
  • ERV2 is another S. cerevisiae helper protein ofinterest for the present invention.
  • Erv2p is a flavin-linked sulfhydryl oxidase localisedto the endoplasmic reticulum lumen, involved in disulphide bond formationwithinthe ER.
  • a published protein sequence forthe proteinErv2p is as follows:
  • ERV2 is encoded by a non-essential gene comprising an ORF that is 0.591 kbp in size, located on chromosome XVI.
  • a published nucleotide coding sequence of ERV2 is as follows, 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: ATGAAACAGATAGTCAAAAGAAGCCATGCCATCAGAATAGTTGCAGCATTAGGAATCATA
  • ERV2 we include fragments or variants thereof having equivalent ERV2-like activity.
  • EUGl is another S. cerevisiae helper protein of interest for the present invention.
  • Euglp is a protein disulphide isomerase of the endoplasmic reticulum lumen, with an overlapping function with Pdilp. It may interact with nascent polypeptides in the ER.
  • a published protein sequence for the protein Euglp is as follows:
  • EUGl is encoded by a non-essential gene comprising an ORF that is 1.554 kbp in size and is located on chromosome IV.
  • a published nucleotide coding sequence of EUGl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidentical proteinproduct:
  • Mpdlp is a member ofthe protein disulphide isomerase (PDI) family. Its over- expression suppresses the defectinmaturation ofcarboxypeptidase Y, and defects in other essential Pdilp functions that can be caused by PDIl deletion.
  • PDI protein disulphide isomerase
  • MPDl is encoded by anon-essential gene comprising an ORF that is 0.957 kbp in size and is located on chromosome XV.
  • a published nucleotide coding sequence ofMPDl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
  • MPDl we include fragments or variants thereof having equivalent MPD 1 -like activity.
  • MPD2 is another S. cerevisiae helper protein of interest for the present invention.
  • Mpd2p is a member of the protein disulphide isomerase (PDI) family. It exhibits chaperone activity. Its overexpression suppresses the lethality of a PDIl deletion but does not complement all Pdilp functions. It undergoes oxidation by Erolp.
  • PDI protein disulphide isomerase
  • MPD2 is encoded by a non-essential gene comprising an ORF that is 0.834kbp in size and is located on chromosome XV.
  • a published nucleotide coding sequence of MPD2 is as follows, 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:
  • MPD2 we include fragments or variants thereof having equivalent MPD2-like activity.
  • Eps Ip is a Pdilp (protein disulphide isomerase)-related protein involved in endoplasmic reticulum retention of resident ER proteins.
  • a published protein sequence for the protein Eps Ip is as follows:
  • EPSl is a non-essential gene comprising an ORF that is 2.106 kbp in size and is located on chromosome DC
  • a published nucleotide coding sequence of EPSl is as follows, 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:
  • 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), is another S. cerevisiae helper protein of interest for the present invention.
  • PDI we include any protein having the ability to reactivate the ribonuclease activity against RNA of scrambled ribonuclease as described in EP 0 746 611 and Hillson et al, 1984, Methods Enzymol, 107, 281-292.
  • PDI is an enzyme which typically catalyses thiobdisulphide 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, ScL, 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 IcD) with characteristically acidic pi (4.0-4.5) (Hillson et 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.
  • Preferred PDI sequences include those from humans and those from yeast species, such as S. cerevisiae.
  • a yeast protein disulphide isomerase precursor, PDIl can be found as Genbank accession no. CAA42373 or BAA00723, It has. the following sequence of 522 ammo acids:
  • yeast protein disulphide isomerase sequence can be found as Genbank accession no. CAA38402. It has the following sequence of 530 amino acids
  • DERI is another S. cerevisiae helper protein of interest for the present invention.
  • Derlp is an endoplasmic reticulum membrane protein, required for the protein degradation process associated with the ER, and is involved in the retrograde transport of misfolded or unassembled proteins.
  • a published protein sequence for the protein Derlp is as follows:
  • DERI is encoded by a non-essential gene comprising an ORF that is 0.636 kbp in size and is located on chromosome II.
  • a published nucleotide coding sequence of DERI is as follows, 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:
  • DER3 is another S. cerevisiae helper protein of interest for the present invention and is also known as HRDl.
  • Der3p is a ubiquitin-protein ligase required for endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins. It is genetically linked to the unfolded protein response (UPR) and is thought to be regulated through association with Hrd3p. It contains an H2 ring finger.
  • a published protein sequence for the protein Der3p is as follows:
  • DER3 is encoded by a non-essential gene comprising an ORF that is 1.656 kbp in size and is located on chromosome XV.
  • a published nucleotide coding sequence of DER3 is as follows, 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:
  • HRD3 is another S. cerevisiae helper protein ofinterest for the present invention.
  • Hrd3p is a resident protein ofthe ER membrane that plays a central role in ER- associated protein degradation (ERAD). It forms an HRD complex with Hrdlp and ERAD determinants that engage in lumen to cytosol communication and coordination of ERAD events.
  • ERAD ER-associated protein degradation
  • Hrd3p is asfollows: MITLLLYLCVICNAIVLIRADSIADPWPEARHLLNT IAK SRDPMKEAAMEPNADEFVGFY VPMDYSPRNEEKNYQSIWQNEITDSQRHIYELLVQSSEQFNNSEATYTLSQIHLWSQYNF PHNMTLAHKYLEKFNDLTHFTNHSAIFDLAVMYATGGCASGNDQTVIPQDSAKALLYYQR AAQLGNLKAKQVLAYKYYSGFNVPRNFHKSLVLYRDIAEQLRKSYSRDEWDIVFPYWESY , NVRISDFESGLLGKGLNSVPSSTVRKRTTRPDIGSPFIAQVNGVQMTLQIEPMGRFAFNG NDGNINGDEDDEDASERRIIRIYYAALNDYKGTYSQSRNCERAKNLLELTYKEFQPHVDN LDPLQVFYYVRCLQLLGHMYFTGEGSSKPNIHMAEEILTTSLEISRR
  • HRD3 is encoded by anon-essential gene comprising an ORF that is 2.502 kbp in size and is located on chromosome XII.
  • a published nucleotide coding sequence ofHKD3 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
  • HRD3 we include fragments or variants thereof having equivalentHRD3-like activity.

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Abstract

The present invention provides a host cell suitable for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over-expression of two or more helper proteins selected from a DnaJ-like protein (such as JEMl), an Hsp70 family protein (such as LHSl) and SILl wherein at least one of the over-expressed two or more helper proteins is selected from JEMl, LHSl and SILl, and wherein the DnaJ-lilce protein is not SCJl.

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.
A key parameter in the development of a commercially viable process for the production of a recombinant protein is the yield of the product from the host organism.
Factors that influence the yield of a particular heterologous protein are complex and include the biochemical and biophysical properties of the protein itself; its influence on, and modification of, the host's own cellular functions; and the choice and deployment of those sequences that are necessary for efficient transcription, translation, secretion (if required) and plasmid stability.
SUMMARY OF THE INVENTION
We have identified a series of proteins (hereinafter "helper" proteins) that are over-expressed in a (non-publicly available) S. cerevisiae that possesses increased production of a protein product of choice, such as a recombinant protein. These over-expressed helper proteins have all, individually, been previously identified.
In the case of some of these helper proteins, there is nothing in the art to suggest that their over-expression would aid in the increased production of a recombinant heterologous protein product of choice. In the case of some of the other identified helper proteins, the (as yet unpublished) art has recognised that their over-expression can aid in increasing the production of a recombinant heterologous protein product of choice (see PCT/GB2004/005462). However, there is nothing in the art to suggest that the combined and simultaneous over-expression of such helper proteins would further enhance the production of a protein product of choice.
Accordingly, the present invention provides a host cell suitable for enhanced production of a protein product of choice wherein the host cell is genetically modified to cause over-expression of one or more of the identified helper proteins.
Thus the present invention provides a host cell that is suitable for enhanced production of a protein product of choice characterised in that the host cell comprises a first gene encoding a first helper protein as defined herein, or a variant thereof, and a second gene encoding a desired protein product of choice, wherein the host cell is genetically modified to cause over-expression of the first helper, protein, and-
(a) wherein the first and second genes are not both present within the host cell on the same 2μm-family plasmid (and optionally the first gene is not present within the host cell on any 2μm-family plasmid; and further optionally the second gene is not present within the host cell on any 2μm- family plasmid); and
(b) wherein the host cell is not genetically modified to cause over-expression of a further helper protein that is different from the first helper protein and is selected from the group consisting of AHAl, CCT2, CCTS, CCT4, CCT5, CCT6, CCTl, CCT8, CNSl, CPR3, CPR6, EROl, EUGl, FMOl, HCHl, HSPlO, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, JEMl, MDJl, MDJ2, MPDl, MPD2, PDIl, PFDl, ABCl, APJl,
ATPIl, ATP 12, BTTl, CDC37, CPRl, HSC82, KAR2, LHSl, MGEl, MRSIl, NOBl, ECMlO, SSAl, SSA2, SSA3, SSA4, SSCl, SSE2, SILl, SLSl, ORMl, ORM2, PERl, PTC2, PSEl, UBU and HACl or a truncated intronless HACl (and optionally, the host cell is not genetically modified to cause over-expression of any further helper protein that is different from the first helper protein).
The thus over-expressed first helper protein may be any helper protein defined below. For example, the over-expressed first helper protein may be a DnaJ-like protein (such as JEMl), an Hsp70 family member protein (such as LHSl) or SILl5 or a variant of any of these. Over-expression of the first helper protein may be achieved by any suitable means of genetic modification known in the art. Suitable examples of such approaches for genetic modification are discussed in more detail below.
The host cell may or may not comprise a recombinant copy, such as a plasmid encoded copy, or a chromosomally integrated recombinant copy, of a gene encoding the further helper protein as defined in (b) above. Thus, in one embodiment, the first helper protein may be the only helper protein that is over- expressed by the host cell.
In another embodiment, the invention provides a host cell that is suitable, for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over-expression of a helper protein selected from the list comprising SCJl, FKB2, SSEl, ERV2, DERl5 DER3, HRD3, UBC7 and D0A4. The host cell may or may not be genetically modified to cause over- expression of two or more helper proteins, at least one of which is a helper protein selected from the list comprising SCJl, FKB2, SSEl, ERV2, DERI, DER3, HRD3, UBC7 and DOA4. In that case, at least one other helper may or may not be selected from the list comprising -
(a) chaperones selected from a DnaJ-like protein (such as JEMl), an
Hsp70 family member protein (such as LHSl), SCJl, KAR2, SILl (note that, SILl has previously been referred to as SLSl), FKB2, SSAl5 SSA2, SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2, ECMlO5 MDJl and MDJ2.
(b) proteins involved in the formation of disulphide bonds in other proteins selected from ERO 1 , ERV25 EUGl , MPD 1 , MPD2, EPS 1 and PDIl;
(c) proteins involved in protein degradation selected from DERI, DER3, HRD3, UBC7 and D0A4; and
(d) HACl.
For example, the host cell may or may not be genetically modified to cause over- expression of two or more helper proteins selected from a DnaJ-like protein (such as JEMl), an Hsp70 family protein (such as LHSl) and SJXl. For example, the host cell according to may or may not be genetically modified to cause over- expression of-
(a) a DnaJ-lilce protein and an Hsp70 family protein; or (b) a DnaJ-like protein and SILl ; or
(c) an Hsp70 family protein and SILl.
The host may or may not be genetically modified to cause over-expression of three or more helper proteins, wherein the three or more helper proteins comprise a DnaJ-like protein, an Hsp70 family protein and SILl, for example JEMl5 LHSl and SILl.
The Hsp70 family protein may or may not be a protein that localises to the lumen of the ER. The Hsp70 family protein may or may not be a prokaryotic Hsp70 family protein. The Hsp70 family protein may or may not be a eukaryotic Hsp70 family protein. The Hsp70 family protein may or may not be LHSl5 KAR2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 or ECMlO, such as from yeast, for example, from S. cerevisiae. LHSl may or may not be a preferred Hsp70 family protein for use in the present invention. Other Hsp70 family proteins for use in the present invention may or may not include a mammalian BiP
(GRP78) (, such as the protein described by Haas and Wabl (1983) Nature 306,
387), a mammalian HSP72 (HSP70), HSP73 (HSC70) or mtp70, a mammalian GRP170 (such as the protein described by Lin et al (1993) MoI. Biol Cell 4,
1109), a mammalian HSP70 protein (such as a protein as reviewed by Ohtsuka and Hata. (2000) International Journal of Hyperthermia 16, 231; Gething and
Sambrook (1992) Nature 355, 33; and/or Craig and Gross (1991) TIBS 16, 135), a
Gallus gάllus HSP70 protein, such as the protein defined by accession number AAO44921 (Mazzi et al (2003) Genet. MoI. Biol. 26, 275-281), a Nicotiana tabacum luminal binding protein (BiP), such as the protein defined by accession number CAA42661 (Denecke et al (1991) Plant Cell 3, 1025), a Paramecium caudatum HSP70 protein, such as the protein defined by accession number
BAEl 6705 (Hori etal (2006) MoI. Phylogenet. Evol. 38, 697), aHordeum vulgar e HSP70 protein, such as a subsp. vulgare HSP70 protein accession number, such as the protein defined by AAA62325 (Chen et al (1994) Plant Physiol. 106, 815), an
Arabidopsis thaliana HSP70 protein accession number NP_187864, the
Chlamydia trachomatis A/HAR-13 chaperone protein dnaK (Heat shock protein
70) (Heat shock 70 kDa protein) (HSP70) , such as the protein defined by accession number Q3KLV7 (Carlson et al (2005) Infect. Immun. 73, 6407), a
Pongo pygmaeus hsp70 protein, such as the protein defined by accession number
CAH92327, a Haemophilus influenzae 86-028NP HSP70 protein, such as the protein defined by accession number YP_249343 (Harrison et al (2005) J
Bacteriol. 187, 4627), a Streptococcus pneumoniae HSP70 protein, such as the protein defined by accession number AAB39221, a Mus musculus HSP70 protein, such as the protein defined by accession number AAC84169 (Xie et al (2003)
Genome Res. 13, 2621), a Bacillus subtilis HSP70 protein, such as the protein defined by accession number BAA12464 (Mizuno et al (1996) Microbiology
(Reading, Engl.) 142, 3103), and a Escherichia coli DnaK protein , such as the protein defined by Slepenkov and Witt (2002) MoI. Microbiol .45, 1197. It will be appreciated that, in the rest of this specification, reference to LHSl may or may not be taken to be, by extension, a reference to an equivalent Hsp70 family protein, such as an Hsp70 family protein as defined in this paragraph. Other preferred Hsp70 family proteins may have an activity equivalent to LHSl, when co-expressed with one or both of JEMl and SlLl, for example in the manner as set out in the present examples. Thus, a host cell of the present invention, when genetically modified to cause simultaneous over-expression of a preferred Hsp70 family protein with one or both of JEMl and SILl, will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl5 the increase being compared to the to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been geneticalfy modified to cause overexpression of any of LHSl, JEMl or SILl.
By "substantially the same increase in the production of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl (the increased being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SJXl).
By "substantially the same reduction of fragmentation of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of LHSl with one or both of JEMl and SILl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SJXl). DnaJ-like proteins are reviewed in Walsh et al, 2004, EMBO reports, 5, 567-571. The DnaJ-like protein typically comprises a J-domain as defined in Walsh et aϊ, 2004, op. cit. the contents of which are incorporated herein by reference. The DnaJ-like protein may or may not be a prokaryotic DnaJ-like protein. The DnaJ- like protein may or may not be a eukaryotic DnaJ-like protein. The DnaJ-like protein may or may not be any one of the yeast DnaJ proteins such as a protein selected from JEMl5 MDJl, MDJ2, SEC63, YDJl5 XDJl5 APJl5 SISl5 DJPl5 ZUOl5 SWA2, JJJl5 JJJ2, JJJ3, CAJl5 CWC23, PAMlS5 JACl, JIDl5 SCJl, HLJl and ERJ5. The DnaJ-like protein may or may not be a protein that localises to the ER5 such as JEMl5 SCJl, HLJl5 SEC63 or ERJ5, and may or may not be a protein that localises to the ER membrane. The DnaJ-like protein may or may not be a protein that localises to the cytoplasm of the host cell, such as YDJl, XDJl, APJl, SISl5 DJPl, ZUOl, SWA2, JJJl5 JJJ2 or JJJ3. The DnaJ-like protein may or may not be a protein that localises to the nucleoplasm of the host cell, such as CAJl or CWC23. The DnaJ-like protein may or may not be a protein that localises to the mitochondria of the host cell, such as MDJl, MDJ2, PAM18, JACl or JlDl. The DnaJ-like protein is typically not SCJl. JEMl may or may not be a preferred DnaJ-like protein for use in the present invention. Other DnaJ-like proteins may or may not include the following proteins or proteins families, or fragments or variants thereof -
• the HSP40 class of proteins (reviewed by Ohtsuka and Hata. (2000) InternationalJournal of Hyperthermia 16, 231 and Table 1 therein);
• a mammalian Erdjl (such as MTJl, Chevalier et al (2000) J Biol. Chem. 275 19620);
• a mammalian Erdj2 such as hSec63, Skowronek et al (1999) J. Biol Chem. 380, 1133);
• a mammalian Erdj3 (such as HEDJ/Scjlp, Shen and Hendershot (2005) MoI. Biol. Cell. 16, 40); • a mammalian Erdj4 (such as described in Shen et al (2002) J Biol. Chem. 277, 15947); • a mammalian Erdj5 (such as described in Cunnea et al (2003) J Biol. Chem. 278, 1059);
• a Gallus gallus DnaJ homolog subfamily B member 11 precursor, such as the ER-associated dnaJ protein 3 ErJ3, the ER-associated Hsp40 co- chaperone (hDj9, or the PWPl -interacting protein 4, such as defined by accession number XP_422682;
• a Nicotiana tabacum DnaJ homolog, such as the protein defined by accession number BAC53943;
• a Arabidopsis thaliana DnaJ homolog, such as the protein defined by accession number AAB49030 (Zhou et al (1999) Plant Physiol. 121,
1053);
• a Chlamydia trachomatis A/HAR-13 Chaperone protein dnaJ, such as the protein defined by accession number YP_328153 (Carlson et al (2005) Infect. Immun. 73, 6407); • a Pongo pygmaeus DnaJ homolog subfamily B member 9, such as the protein defined by accession number Q5R9A4;
• a Haemophilus influenzae Rd KW20 Dna-J like membrane chaperone protein, such as the protein defined by accession number NP_438440 (Fleischmann et al (1995) Science 269, 496); • a Escherichia coli DnaJ protein, such as the protein defined by accession number AAA00009 (Ohki et al (1986) J. Biol. Chem. 261, 1778);
• a Escherichia coli DnaJ-like protein, such as the protein defined by accession number BAB96590 (Musso et al (1977) Proc. Natl. Acad. Sd. U.S.A. 74, 106); • a Streptococcus pneumoniae DnaJ protein, such as the protein defined by accession number AAB39222;
• a Mus musculus DnaJ homolog, such as a subfamily B member 6 (Heat shock protein J2) (HSJ-2) (MRJ) (mDj4) , such as the protein defined by accession number XP_987742; • a Bacillus subtilis DnaJ protein, such as the protein defined by accession number BAA12465 (Mizuno et al (1996) Microbiology (Reading, Engl.) 142, 3103); and • a plant Sorghum bicolour DNAJ domain protein, such as the protein defined by accession number ABF48023.
It will be appreciated that, in the rest of this specification, reference to JEMl may or may not be taken to be, by extension, a reference to an equivalent DnaJ-like protein, such as a DnaJ-like protein as defined in the above paragraph.
Other preferred DnaJ-like proteins may have an activity equivalent to JEMl, when co-expressed with one or both of LHSl and SILl, for example in the manner as set out in the present examples. Thus, a host cell of the present invention, when genetically modified to cause simultaneous over-expression of a preferred DnaJ- like protein with one or both of LHSl and SILl, will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of JEMl with one or both of LHSl and SILl, the increase being compared to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl.
By "substantially the same increase in the production of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase, in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of JEMl with one or both of LHSl and SILl (the increase being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
By "substantially the same reduction of fragmentation of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of JEMl with one or both of LHS 1 and SILl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
The host cell that is genetically modified to cause over-expression of two or more, such as at least three, helper proteins selected from a DnaJ-like protein, an Hsp70 family protein and SILl may or may not be further genetically modified to cause over-expression of at least one, two, three, four, five, six or seven proteins involved in the formation of disulphide bonds in other proteins selected from the group consisting of EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl. PDIl may or may not be preferred.
In another embodiment, the invention provides a host cell suitable for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over-expression of three or more helper proteins, wherein the three or more' helper proteins are selected from the list comprising —
(a) chaperones selected from a DnaJ-like protein (such as JEMl), an
Hsρ70 family member protein (such as LHSl)5 SCJl, KAR2, SILl, FKB2, SSAl, SSAV SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2, ■ ECMl O5 MDJl and MDJ2.
(b) proteins involved in the formation of disulphide bonds in other proteins selected from EROl, ERV2, EUGl5 MPDl, MPD2, EPSl and PDIl;
(c) proteins involved in protein degradation selected from DERI, ■ DER3, HRD3, UBC7 and D0A4; and
(d) HACl. The three or more helper proteins may or may not comprise at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or seventeen of the chaperones selected from the group consisting of JEMl5 an Hsp70 family member protein (such as LHSl)5 SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl and MDJ2. The three or more helper proteins may or may not comprise at least one, two, three, four, five, six or seven proteins involved in the formation of disulphide bonds in other proteins selected from the group consisting of EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl. The three or more helper proteins may or may not comprise at least one, two, three, four or five of the proteins involved in protein degradation selected from DERI, DER3, HRD3, UBC7 and DOA4.
It will be appreciated that the host cell may or may not comprise a polynucleotide sequence that encodes a protein product of choice.
In one embodiment, the host cell comprises a polynucleotide sequence that encodes a protein product of choice. The protein product of choice may or may not be a protein that is naturally produced by the host cell or may or may not be a heterologous protein. In this context, a "heterologous protein" is a protein that is not naturally encoded by the host cell. The polynucleotide sequence that encodes the protein product of choice may or may not be an endogenous polynucleotide sequence or (in particular, where the protein product of choice is a heterologous protein) the polynucleotide sequence that encodes the protein product of choice may or may not be an exogenous polynucleotide, and the exogenous polynucleotide may or may not be integrated into the chromosome of the host cell or present in the host cell as part of a replicable vector, such as a plasmid.
However, the present invention also contemplates the production of host cells suitable for enhanced production of a protein product of choice, into which an appropriate polynucleotide sequence, encoding the protein product of choice, can be later introduced. Therefore, in another embodiment, the host cell does not comprise a polynucleotide sequence that encodes a protein product of choice. Suitable host cells are discussed below.
By "enhanced production" we include the meaning that the level of production of protein product of choice is greater in a cultured population of the genetically modified host cell than in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins. Typically, the measurement can be made under culture conditions that are standard for the growth of the host cell that is being used.
Thus the production of the protein product of choice in a cultured population of the genetically modified host cell of the invention be greater than, typically at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% (i.e. 1.1-fold), 20% (i.e. 1.2-fold), 30% (Le. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. l:8-fold), 90% (i.e. 1.9-fold), 100% (i.e. 2-fold), 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold5 30-fold, 40- fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold greater than, the production of the protein product of choice in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins. These figures may, or may not, be figures that have been normalised to account for differences in the cell growth of the two cultured populations, as compared.
For example, the production of the protein product of choice in a cultured population of the genetically modified host cell of the invention may be up to 10% (Le. 1.1-fold), 2O°/o (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (Le. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9-fold), 100% (Le. 2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- • fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800- fold, 900-fold, 1000-fold or 2000-fold greater than the production of the protein product of choice in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins. These figures may, or may not, be figures that have been normalised to account for differences in the cell growth of the two cultured populations, as compared.
Typically, the protein product of choice may be produced in a cultured population of the genetically modified host cell of the invention to produce a culture containing at least 0.001 g.L"1, such as at least 0.01 g.L"1, at least 0.1 g.L"1, 1 g.L"1, 2 g.L"1, 3 g.1/1, 4 g.1/1, 5 g.1/1, 6 g.1/1, 7 g.1/1, 8 g.1/1, 9 g.L/1, 10 g.L- 1, 20 g.1/1, 30 g.L/1, 40 g.L"1, 50 g.L"1, 60 g.L/1, 70 g.L/1, 80 g.L4, 90 g.L"1, or 100 g.L"1 of the protein product of choice. The protein product of choice may be produced in a cultured population of the genetically modified host cell of the invention to produce a culture containing up to 0.01 g.L"1, 0.1 g.L"1, 1 g.L"1, 2 g.L"1, 3 g.L"1, 4 g.L"1, 5 g.L"1, 6 g.1/1, 7 g.1/1, 8 g.L"1, 9 g.L"1, 10 g.L"1, 20 g.L"1, 30 g.L"1, 40 g.1/1, 50 g.L"1, 60 g.1/1, 70 g.L"1, 80 g.L"1, 90 g.L'1, 100 g.L"1 or 200 g.L"1 of the protein product of choice.
By "enhanced production" we also include the meaning that the level of activity of the protein product of choice that is produced by the host cell is greater in a cultured population of the genetically modified host cell than in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins. The nature of the activity will depend on the identity of the protein product of choice and may, for example, be a measurement of the catalytic activity of the protein upon a substrate or the binding properties of rtie protein to a ligand. Typically, the measurement of protein activity can be made under culture conditions that are standard for the growth of the host cell that is being used or following isolation of the. protein from the culture medium. Ln either case, the comparison should be made on the basis of activity per unit volume of culture or protein recovered . therefrom. The comparison may, or may not, be normalised to account for differences in the cell growth of the two cultured populations, as compared.
Thus the activity of the protein product of choice that is produced in a cultured population of the genetically modified host cell of the invention may be greater than, typically at least 10% (i.e. 1.1-fold), 20% (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5-fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7-fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9-fold), 100% (i.e. 2-fold), 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70- fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 104-fold, 105-fold, or 106-fold greater than, the activity of the protein product of choice in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins.
For example, the activity of the protein product of choice in a cultured population of the genetically modified host cell of the invention may be up to 10% (i.e. 1.1- fold), 20% (i.e. 1.2-fold), 30% (i.e. 1.3-fold), 40% (i.e. 1.4-fold), 50% (i.e. 1.5- fold), 60% (i.e. 1.6-fold), 70% (i.e. 1.7 fold), 80% (i.e. 1.8-fold), 90% (i.e. 1.9- fold), 100% (Le. 2-fold), 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 104-fold, 105-fold, or 106-fold greater than the activity of the protein product of choice in a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins.
By "enhanced production" we include the additional or alternative meaning that the level of degradation of the protein product of choice is reduced when produced by a cultured population of the genetically modified host cell of the present invention compared to the level of degradation of the protein product of choice when produced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention. The level of protein degradation can be determined by quantification of fragments of the protein product of choice relative to the total of the protein product of choice, for example when by analysis of SDS-PAGE using densitometry. When expressed as a percentage of detected protein product fragments. relative to total protein product levels detected (i.e. total protein product detected = full length protein product + degradation products) then the percentage of detected protein product fragments when produced by a cultured population of the genetically modified host cell of the present invention may be, or be less than, 99%, 98%, 97%, 96%, 05%, 04%, 03%, 92%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less, such as up to 98%, 97%, 96%, 95%, 94%, 93%, 92%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the percentage of detected protein product fragments when produced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention. These values may or may not be normalised, for example based on culture optical density readings, to account for different growth rates observed between strains.
By "enhanced production" we include the additional or alternative meaning that the level of post-translational modification of the protein product of choice is increased or reduced when produced by a cultured population of the genetically modified host cell of the present invention compared to the level of post- translational modification of the protein product of choice when produced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention. For example, the altered (i.e. increased or reduced) level of post-translational modification may be an alteration in the level of proteolytic cleavage, hexosylation (for example mannosylation), glycosylation, phosphorylation, phosphopantetheinylation, carbamylation, carboxylation (such as γ-carboxylation), sialation, sulphonation, hydroxylation, prenylation, isoprenylation, acylation, ubiquitination, lipoylation, biotinylation, glycylation, glutamylation, methylation, . alkylation, acetylation, formylation, selenation, disulphide bond formation or oligomerisation of the protein product of choice . The level of post-translational modification of the protein product of choice can be determined by methods well known in the art, such as by mass spectrometry techniques (for example, see Larsen et al, 2006, BioTechniques, 40, 790-798) well known in the art.
By "enhanced production" we include the additional or alternative meaning that the level of stress experienced by a cell that is being cultured to produce the protein product of choice is reduced, compared to the level of stress experienced by a cultured population of the same host cell that has not been genetically modified to cause over-expression of one or more of the identified helper proteins according to the present invention. For example, "enhanced production" can include the additional or alternative meaning that the unfolded protein response is reduced in a host cell. The level of stress, and the level of the unfolded protein response, can be measured by determination of the proportion of HACl1 to total HACl transcript levels. Total HACl transcript levels are the sum of HACl1 transcript levels and unspliced HACl (HACl") transcript levels in a cell. A reduced proportion of HACl1 transcript levels compared to total HACl transcript level, relative to a control, is indicative of reduced stress and reduced UPR signalling relative to that control. Helper proteins suitable for achieving this effect may include Hsp70 family proteins (such as LHSl) and DnaJ-like proteins (such as JEMl) and combinations of other helper proteins such as disclosed in the present application.
In principle, any "protein product of choice" can be produced. The identity of preferred embodiments of the "protein product of choice" is discussed further below.
The host cell is genetically modified to cause over-expression of one or more of the helper proteins. By "over-expression", in the context of helper proteins, we mean that the measurable level of mRNA encoding the one or more helper proteins, and/or the measurable level of the one or more helper proteins themselves, and/or the measurable level of the helper protein activity, is greater than the measurable level in a host cell that has not been genetically modified. Typically, the measurement will be made under culture conditions that are standard for the growth of the host cell that is being used. Standard conditions for yeast cell growth are discussed, for example, in WO 96/37515, WO 00/44772 and WO 99/00504, the contents of which are incorporated herein by reference.
Thus the host cell may or may not be genetically modified to cause a level of expression of one or more of the helper proteins that is at least a 1.1 -fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or more, of the unmodified level of expression of one or more of the helper proteins.
For example, the host cell may or may not be genetically modified to cause a level of expression of one or more of the helper proteins that is up to 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or 100-fold of the unmodified -type level of expression of one or more of the helper proteins.
For example, the host cell may be genetically modified to cause a level of expression of one or more of the helper proteins that is between 1- to 30-fold, such as about 2- to 25-fold, of the unmodified-type level of expression of one or more of the helper proteins.
The host cell may or may not be genetically modified to cause over-expression of one or more of the helper proteins by the introduction of one or more recombinant copies of one or more polynucleotides that each comprise a region (the "coding region", or "open reading frame", which can be abbreviated to "ORF") that encodes one or more helper proteins.
A copy of the polynucleotide may or may not be introduced into the chromosome of the host cell and/or may or may not be encoded by a plasmid or other vector that is used to transform the host cell. The polynucleotide may or may not comprise some or all of the regulatory sequences necessary to cause transcription and/or translation of the ORP of the polynucleotide.
Regulatory sequences necessary to cause transcription and/or translation of the ORF of the polynucleotide include sequences that modulate (i.e., promotes or reduces, typically promotes) 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 be about, or up to, 5bp, lObp, 15bp, 20bp, 25bp, 30bp, 35bp, 40bp, 45bp, 50bp, 60bp, 70bp, 80bp, 90bp, lOObp, 120bp, 140bp, 160bp, 180bp, 200bp, 220bp, 240bp, 260bp, 280bp, 300bp, 35Obp, 400bρ, 450bp, 500bρ, 550bp, 60,0bp, 650bp, 700bρ, 750bp, 800bp, 850bρ, 900bp, 950bρ, lOOObp, HOObp, 1200bp, 1300bp, 1400bp, 1500bp or greater, in length. '
Such non-coding regions and regulatory regions are not restricted to the native non-coding regions and/or regulatory regions naturally associated with the ORF.
Where the host cell is yeast, such as Saccharomyces cerevisiae, suitable promoters for S. cerevisiae include those associated with the PGKl gene, GALl or GALlO genes, TEFl, TEF2, PYKl, PMAl, CYCl, PH05, TRPl, ADHl, ADH2, the genes for glyceraldehyde-3 -phosphate dehydrogenase (for example, TDHl, TDH2 or
TDH3), hexokinase (for example, HXKl or HXK2), pyruvate decarboxylase (for example, PDCl, PDC5 or PDC6), phosphofructokinase (for example, PFKl or PFKl), triose phosphate isomerase (for example, TPIl), phosphoglucose isomerase (for example, PGIl), glucokinase (for example, GLKl), α-mating factor pheromone (for example, MFa-I or MFa-2), a-mating factor pheromone (for example, MFAl or MFA2), PRBl, PRAl, GPDl, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5' regulatory regions of other promoters or with upstream activation sites (e.g. the promoter of EP-A-258 067). Where multiple ORFs are to be expressed, a different promoter may or may not be chosen for each ORF. The skilled person can readily determine appropriate combinations of promoters. For example, the promoters from the ADHl, PGKl, TDHl and TEFl genes are used in combination to recombinantly over-express four helper proteins in Example 3 below.
Suitable transcription termination signals are well known in the art. Where the host cell is eukaryotic, the transcription termination signal is preferably derived from the 3' flanking sequence of a eukaryotic gene, which contains proper signals for transcription termination and polyadenylation. Suitable 3' flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, i.e. may correspond to the promoter. Alternatively, they may be different. Li that case, and where the host is a yeast, preferably S. cerevisiae, then the termination signal of the & cerevisiae ADHl, ADH2, CYCl, or PGKl genes are preferred.
It may be beneficial for the promoter and open reading frame 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, and visa versa.
In one embodiment, a suitable regulatory sequences in yeast, such as Saccharomyces cerevisiae, includes: a yeast promoter (e.g. the Saccharomyces cerevisiae PRBl promoter), as taught in EP 431 880; and a transcription terminator, preferably the terminator from Saccharomyces ADHl, as taught in EP 60 057. Other suitable regulatory sequences are given in. the examples, and include TEFl, PGKl and TDHl promoters. It may be beneficial for the non-coding region to incorporate more than one DNA sequence encoding a translational stop codon, such as UAA5 UAG or UGA5 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. Preferably, the polynucleotide incorporates at least two translation stop codons.
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 ORF in its intended manner. Thus a regulatory region "operably linked" to an ORF is positioned in such a way that the regulatory region is able to influence transcription and/or translation of the ORF in the intended manner, under conditions compatible with the regulatory sequence.
Alternatively, the polynucleotide may or may not be formed in such a manner that it can take advantage of endogenous regulatory sequences within the chromosome or plasmid to cause transcription and/or translation of the coding region of the polynucleotide. For example, the use of promoterless constructs is well known in the art as a way of allowing an endogenous promoter sequence to drive the expression of a recombinantly-introduced polynucleotide coding region.
The skilled person will appreciate that the host cell may or may not comprise endogenous copies of genes encoding one or more of the helper proteins. Therefore, this invention also contemplates genetic modifications to the host cell that cause increased steady state levels of mRNA molecules encoding one or more helper proteins and/or increased steady state levels of one or more helper proteins.
This can include the genetic modification of operably linked endogenous regulatory regions. For example, the endogenous promoter in the gene of an endogenously encoded helper protein can be replaced by a promoter that causes greater levels of expression of the helper protein under culture conditions. Alternatively, genetic modifications can be made to cis or trans regulators of the gene of an endogenously encoded helper protein, so as to increase the expression of the helper protein under culture conditions. Thus, the polynucleotide region that encodes a genetically encoded repressor of a gene of an endogenously encoded helper protein could be genetically modified to reduce or prevent repression of the endogenous helper protein gene.
Alternative genetic modifications to increase the expression of a helper protein or protein product of choice can involve transient expression techniques known in the art. For example, suitable techniques are disclosed in Chen et al, 1997, Nucleic Acids Research, 25, 4416-4418 and in Behr et al, 1989, Proc. Natl. Acad. Sci. USA, 86, 6982-6986.
Thus, a number of techniques are available to the skilled person to genetically modify a cell to cause over-expression of a helper protein (and the same techniques may be used to cause expression of a protein product of choice). Suitable techniques include -
(i) introduction of a recombinant copy of an encoding polynucleotide by integration into the chromosome of the host cell (for example, either
■with associated regulatory sequences or without associated regulatory sequences so as to take advantage of endogenous regulatory sequences at the site of integration);
(ii) introduction of plasmid or other vector comprising a recombinant copy of an encoding polynucleotide into the cell;
(iii) genetic modifications of a host cell's endogenous regulatory region operably linked to the host cell's endogenous copy of an ORF encoding a helper protein or protein product of choice, to cause increased steady state levels of mRNA molecules encoded by said • ORF;
(iv) genetic modifications to a cis or trans regulator of the gene of an endogenously encoded helper protein or protein product of choice ; or
(v) transient expression of a helper protein or protein product of choice. Where the host cell comprises a first gene encoding a protein product of choice, and a second gene encoding a first helper protein, then for example,
• the first gene may be a gene as defined in (i) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (ii) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the first and second genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the second gene);
• the first gene may be a gene as defined in (iii) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iv) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; or • the first gene may be a gene as defined in (v) above and the second gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above.
Where the host cell comprises a first gene encoding a protein product of choice, and a second gene encoding a first helper protein and a third gene encoding a second helper protein, then for example,
• the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
• the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene4 as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the first and third genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the third gene);
• the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where the first, second and third genes are introduced on plasmid or vector, the first gene may or may not be introduced on the same plasmid or vector as the second gene, the first gene may or may not be introduced on the same plasmid or vector as the third gene and the second gene may or may not be introduced on the same plasmid or vector as the third gene); • the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (ii) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (i) above,- and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
• the first gene may be a gene as defined in (i) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iii) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
• the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as. defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (iv) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (i) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (ii) above, and the third gene may be a gene as defined in (i)5 (ii), (iii), (iv) or (v) above (and where both the second and third genes are introduced on plasmid or vector, the second gene may or may not be introduced on the same plasmid or vector as the third gene);
• the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (iii) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above;
• the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (iv) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above; or • the first gene may be a gene as defined in (v) above, and the second gene may be a gene as defined in (v) above, and the third gene may be a gene as defined in (i), (ii), (iii), (iv) or (v) above.
Further combinations of possible genetic modifications will be apparent to the skilled person, in light of the above disclosure, when further genes (for example a fourth gene encoding a third helper protein; a fifth gene encoding a fourth helper protein, etc.) are to be over-expressed in the host cell of the invention.
The skilled person can readily choose the most appropriate and convenient method to achieve over-expression of one or more helper proteins in a host cell. It will be appreciated that, in the case that multiple helper proteins are over-expressed in the host cell, at least one helper protein may or may not be over-expressed by the introduction of an appropriate recombinant polynucleotide sequence as discussed above, whereas at least one other helper protein may or may not be over-expressed by a genetic modification to the host cell to cause over-expression of the helper protein from the endogenous gene that encodes it. HELPER PROTEINS
As discussed above, we have identified a series of proteins (hereinafter "helper" proteins) that are over-expressed in a S. cerevisiae strain identified as possessing increased production of a recombinant protein. These over-expressed helper proteins have all, individually, been previously identified.
The helper proteins identified include proteins that can be categorised as follows -
(i) chaperones,
(ii) proteins involved in disulphide bond formation,
(iii) proteins involved in the protein degradation pathway, and
(iv) HACl (encoded by a spliced or unspliced polynucleotide).
These groups are individually described further below.
Chaperones
The class of proteins known as chaperones have been defined by Hartl (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.
For the purposes of the present invention, chaperones of interest can be broadly split into the following three functional sub-groups -
• ER luminal localised chaperones;
• Chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation; and
• Mitochondrial chaperone and translocation proteins Each of these groups are discussed in more detail below.
ER luminal localised chaperones
ER luminal localised chaperones, involved in "protein folding" include DnaJ-like proteins (such as JEMl), Hsp70 family member proteins (such as LHSl), SCJl, KAR2, SILl and FKB2. A detailed description of these proteins and their genes is given separately below.
In one embodiment, the host cell may or may not be genetically modified to cause over-expression of one, or more, of the above ER luminal localised chaperones. For example, SCJl may or may not be over-expressed. Alternatively, FKB2 may or may not be over-expressed.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of two of the above ER luminal localised chaperones. For example, one of the folio wing combinations may or may not be chosen -
• A DnaJ-like proteins (such as JEMl) in combination with one of an Hsp70 family member protein (such as LHS 1), SCJl , KAR2, SILl or FKB2;
• An Hsp70 family member protein (such as LHS 1) in combination with one of SCJl, KAR2, SILl or FKB2;
• SCJl in combination with one of KAR2, SILl or FKB2;
• KAR2 in combination with one of SILl or FKB2; or • SILl in combination with FKB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of three of the above ER luminal localised chaperones. For example, one of the following combinations may or may not be chosen -
JEMl5 LHSl and SCJl; JEMl, LHSl and KAR2; JEMl, LHSl and SILl; JEMl, LHSl and FKB2; JEMl, SCJl and KAR2; JEMl, SCJl and SILl; JEMl, SCJl and FKB2; JEMl5 KAR2 and SILl; JEMl, KAR2 and FKB2; JEMl5 SILl and FKB2; LHSl5 SCJl and KAR2; LHSl5 SCJl and SILl; LHSl5 SCJl and FKB2; LHSl5 KAR2 and SILl; LHSl, KAR2 and FKB2; LHSl5 SILl and FKB2; SCJl, KAR2 and SILl; SCJl5 KAR2 and FKB2; SCJl5 SILl and FKB2; or KAR2, SILl and FKB2.
In one embodiment, the host cell may or may not be genetically modified to cause over-expression of four- of the above ER luminal localised chaperones. For example, one of the following combinations may or may not be chosen -
JEMl, LHSl5 SCJl and KAR2; JEMl5 LHSl5 SCJl and SILl; JEMl, LHSl5 SCJl and FKB2; JEMl5 LHSl5 KAR2 and SILl; JEMl, LHSl5 KAR2 and FKB2; JEMl5 LHSl5 SILl and FKB2; JEMl5 SCJl, KAR2 and SILl; JEMl, SCJl5 KAR2 and FKB2; JEMl, SCJl5 SILl and FKB2; JEMl5 KAR2, SILl and FKB2; LHSl, SCJl5 KAR2 and SILl; LHSl, SCJl5 KAR2 and FKB2; LHSl, SCJl, SILl and FKB2; LHSl, KAR2, SILl and FKB2; or SCJl, KAR2, SILl and FKB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of five of the above ER luminal localised chaperones. For example, one of the following combinations may or may not be chosen -
JEMl5 LHSl5 SCJl5 KAR2 and SILl; JEMl, LHSl, SCJl, KAR2 and FKB2; JEMl, LHSl, SCJl, SILl and FKB2; JEMl5 LHSl5 KAR2, SILl and FKB2; JEMl5 SCJl, KAR2, SJJLl and FKB2; or LHSl, SCJl, KAR2, SJJLl and FK32.
In another embodiment, the host cell may or may not be genetically modified to - cause over-expression of all six of the above. ER luminal localised chaperones. In other words, the following combination may or may not be chosen -
JEMl5 LHSl5 SCJl, KAR2, SILl and FKB2. In one preferred embodiment, the host cell may or may not be genetically modified to cause over-expression of two, three or four helper proteins selected from LHSl, SILl5 JEMl and SCJl5 such as one of the following combinations -
LHSl and SILl; LHSl and JEMl; LHSl and SCJl; SILl and JEMl; SILl and SCJl; JEMl and SCJl; LHSl, SILl and JEMl; LHSl5 SILl and SCJl; LHSl5 JEMl and SCJl; SILl5 JEMl and SCJl; or LHSl5 SILl5 JEMl and SCJl.
Chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation
Chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation include SSAl5 SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl5 SSB2. A detailed description of these proteins and their genes is given separately below.
In one embodiment, rtie host cell may or may not be genetically modified to cause over-expression of one of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, SSEl may or may not be chosen.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of two of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen -
SSAl in combination with one of SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl5 SSB2; SSA2 in combination with one of SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2; SSA3 in combination with one of SSA45 SSEl5 SSE2, SSBl5 SSB2; ■ SSA4 in combination with one of SSEl5 SSE2, SSBl5 SSB2; SSEl in combination with one of SSE2, SSBl5 SSB2;
SSE2 in combination with one of SSBl, SSB2; or SSBl in combination with SSB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of three of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen -
SSAl, SSA2 and SSA3; SSAl, SSA2 and SSA4; SSAl, SSA2 and SSEl; SSAl, SSA2 and SSE2; SSAl, SSA2 and SSBl; SSAl, SSA2 and SSB2; SSAl, SSA3 and SSA4; SSAl, SSA3 and SSEl; SSAl5 SSA3 and SSE2; SSAl, SSA3 and SSBl; SSAl, SSA3 and SSB2; SSAl, SSA4 and SSEl; SSAl, SSA4 and SSE2; SSAl5 SSA4 and SSBl; SSAl, SSA4 and SSB2; SSAl5 SSEl and SSE2; SSAl5 SSEl and SSBl; SSAl5 SSEl and SSB2; SSAl, SSE2 and SSBl; SSAl5 SSE2 and SSB2; SSAl5 SSBl and SSB2; SSA2, SSA3 and SSA4; SSA2, SSA3 and SSEl; SSA2, SSA3 and SSE2; SSA2, SSA3 and SSBl; SSA2, SSA3 and SSB2; SSA2, SSA4 and SSEl; SSA2, SSA4 and SSE2; SSA2, SSA4 and SSBl; SSA2, SSA4 and SSB2; SSA2, SSEl and SSE2; SSA2, SSEl and SSBl; SSA2, SSEl and SSB2; SSA2, SSE2 and SSBl; SSA2, SSE2 and SSB2; SSA2, SSBl and SSB2; SSA3, SSA4 and SSEl; SSA3, SSA4 and SSE2; SSA35 SSA4 and SSBl; SSA3, SSA4 and SSB2; SSA3, SSEl and SSE2; SSA3, SSEl and SSBl; SSA3, SSEl and SSB2; SSA3, SSE2 and SSBl; SSA3, SSE2 and SSB2; SSA3, SSBl and SSB2; SSA4, SSEl and SSE2; SSA4, SSEl and SSBl; SSA4, SSEl and SSB2; SSA4, SSE2 and SSBl; SSA4, SSE2 and SSB2; SSA4, SSBl and SSB2;' SSEl5 SSE2 and SSBl; SSEl, SSE2 and SSB2; SSEl, SSBl and SSB2; or SSE2, SSBl and SSB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of four of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen — SSAl, SSA2, SSA3 and SSA4; SSAl5 SSA2, SSA3 and SSEl; SSAl, SSA2, SSA3 and SSE2; SSAl5 SSA2, SSA3 and SSBl; SSAl, SSA2, SSA3 and SSB2; SSAl, SSA25 SSA4 and SSEl; SSAl5 SSA2, SSA4 and SSE2; SSAl, SSA2, SSA4 and SSBl; SSAl, SSA2, SSA4 and SSB2; SSAl5 SSA2, SSEl and SSE2; SSAl, SSA2, SSEl and SSBl; SSAl5 SSA2, SSEl and SSB2; SSAl, SSA25 SSE2 and SSBl; SSAl5 SSA2, SSE2 and SSB2; SSAl5 SSA2, SSBl and SSB2; SSAl, SSA3, SSA4 and SSEl; SSAl, SSA3, SSA4 and SSE2; SSAl, SSA3, SSA4 and SSBl; SSAl, SSA3, SSA4 and SSB2; SSAl, SSA3, SSEl and SSE2; SSAl, SSA3, SSEl and. SSBl; SSAl5 SSA3, SSEl and SSB2; SSAl, SSA3, SSE2 and SSBl; SSAl, SSA3, SSE2 and SSB2; SSAl, SSA3, SSBl and SSB2; SSAl, SSA4, SSEl and SSE2; SSAl, SSA4, SSEl and SSBl; SSAl, SSA4, SSEl and SSB2; SSAl, SSA4, SSE2 and SSBl; SSAl, SSA4, SSE2 and SSB2; SSAl, SSA4, SSBl and SSB2; SSAl, SSEl, SSE2 and SSBl; SSAl, SSEl5 SSE2 and SSB2; SSAl, SSEl, SSBl and SSB2; SSAl5 SSE2, SSBl and SSB2; SSA2, SSA3, SSA4 and SSEl; SSA2, SSA35 SSA4 and SSE2; SSA2, SSA3, SSA4 and SSBl; SSA2, SSA3, SSA4 and SSB2; SSA2, SSA3, SSEl and SSE2; SSA2, SSA3, SSEl and SSBl; SSA2, SSA3, SSEl and SSB2; SSA2, SSA3, SSE2 and SSBl; SSA2, SSA3, SSE2 and SSB2; SSA2, SSA3, SSBl and SSB2; SSA2, SSA4, SSEl and SSE2; SSA2, SSA4, SSEl and SSBl; SSA2, SSA4, SSEl and SSB2; SSA2, SSA4, SSE2 and SSBl; SSA2, SSA4, SSE2 and SSB2; SSA2, SSA4, SSBl and SSB2; SSA2, SSEl, SSE2 and SSBl; SSA2, SSEl, SSE2 and SSB2; SSA2, SSEl, SSBl and SSB2; SSA2, SSE2, SSBl and SSB2; SSA3, SSA4, SSEl and SSE2; SSA3, SSA4, SSEl and SSBl; SSA3, SSA4, SSEl and SSB2; SSA3, SSA4, SSE2 and SSBl; SSA3, SSA4, SSE2 and SSB2; SSA3, SSA4, SSBl and SSB2; SSA3, SSEl, SSE2 and SSBl; SSA3, SSEl5 SSE2 and SSB2; SSA3,- SSEl5 SSBl and SSB2; SSA3, SSE2, SSBl and SSB2;.SSA4, SSEl, SSE2 and SSBl; SSA4, SSEl, SSE2 and SSB2; SSA4, SSEl, SSBl and SSB2; SSA4, SSE2, SSBl and SSB2; orSSEl, SSE2, SSBl andSSB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of five of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen -
SSAl, SSA2, SSA3, SSA4 and SSEl; SSAl, SSA2, SSA3, SSA4 and SSE2; SSAl, SSA2, SSA3, SSA4 and SSBl; SSAl5 SSA2, SSA3, SSA4 and SSB2;
SSAl, SSA2, SSA3, SSEl and SSE2; SSAl, SSA2, SSA3, SSEl and SSBl;
SSAl5 SSA2, SSA3, SSEl and SSB2; SSAl, SSA2, SSA3, SSE2 and SSBl;
SSAl, SSA2, SSA3, SSE2 and SSB2; SSAl5 SSA2, SSA3, SSBl and SSB2;
SSAl, SSA2, SSA4, SSEl and SSE2; SSAl, SSA2, SSA4, SSEl and SSBl; SSAl, SSA2, SSA4, SSEl and SSB2; SSAl, SSA2, SSA4, SSE2 and SSBl;
SSAl, SSA2, SSA4, SSE2 and SSB2; SSAl, SSA2, SSA4, SSBl and SSB2; . SSAl5 SSA2, SSEl5 SSE2 and SSBl; SSAl, SSA2, SSEl, SSE2 and SSB2;
SSAl, SSA2, SSEl5 SSBl and SSB2; SSAl, SSA2, SSE2, SSBl and SSB2;
SSAl, SSA3, SSA4, SSEl and SSE2; SSAl, SSA3, SSA4, SSEl and SSBl; SSAl, SSA3, SSA4, SSEl and SSB2; SSAl, SSA3, SSA4, SSE2 and SSBl;
SSAl, SSA3, SSA4, SSE2 and SSB2; SSAl5 SSA3, SSA4, SSBl and SSB2;
SSAl, SSA3, SSEl5 SSE2 and SSBl; SSAl5 SSA3, SSEl, SSE2 and SSB2;
SSAl, SSA3, SSEl5 SSBl and SSB2; SSAl5 SSA3, SSE2, SSBl and SSB2;
SSAl5 SSA4, SSEl5 SSE2 and SSBl; SSAl5 SSA45 SSEl5. SSE2 and SSB2; SSAl, SSA4, SSEl5 SSBl and SSB2; SSAl5 SSEl5 SSE2, SSBl and SSB2;
SSA2, SSA3, SSA4, SSEl and SSE2; SSA2, SSA3, SSA45 SSEl and SSBl;
SSA2, SSA3, SSA4, SSEl and SSB2; SSA2, SSA3, SSA4, SSE2 and SSBl;
SSA2, SSA3, SSA4, SSE2 and SSB2; SSA2, SSA3, SSA4, SSBl and SSB2;
SSA2, SSA35.SSEl5 SSE2 and SSBl; SSA2, SSA3,. SSEl, SSE2 and SSB2; SSA2, SSA3, SSEl5 SSBl and SSB2; SSA25 SSA3, SSE2, SSBl and SSB2;
SSA2, SSA4, SSEl, SSE2 and SSBl; SSA2, SSA45 SSEl, SSE2 and SSB2;
SSA2, SSA4, SSEl, SSBl and SSB2; SSA2, SSA4, SSE2, SSBl and SSB2;
SSA25 SSEl5 SSE2, SSBL and SSB2; SSA3, SSA4, SSEl, SSE2 and SSBl;
SSA3, SSA4, SSEl5 SSE2 and SSB2; SSA3, SSA4, SSEl5 SSBl and SSB2; ■ SSA35 SSA4, SSE2, SSBl and SSB2; SSA3, SSEl5 SSE2, SSBl and SSB2; or
SSA45 SSEl5 SSE2, SSBl and SSB2. In another embodiment, the host cell may or may not be genetically modified to cause over-expression of six of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen -
SSAl, SSA2, SSA3, SSA4, SSEl and SSE2; SSAl5 SSA2, SSA3, SSA4, SSEl and SSBl; SSAl, SSA2, SSA3, SSA4, SSEl and SSB2; SSAl, SSA2, SSA3, SSA4, SSE2 and SSBl; SSAl, SSA2, SSA3, SSA4, SSE2 and SSB2; SSAl, SSA2, SSA3, SSA4, SSBl and SSB2; SSAl5 SSA2, SSA3, SSEl5 SSE2 and SSBl; SSAl, SSA2, SSA3, SSEl5 SSE2 and SSB2; SSAl5 SSA2, SSA35 SSEl5 SSBl and SSB2; SSAl5 SSA25 SSA3, SSE2, SSBl and SSB2; SSAl5 SSA2, SSA4, SSEl5 SSE2 and SSBl; SSAl5 SSA2, SSA4, SSEl5 SSE2 and SSB2; SSAl5 SSA2, SSA4, SSEl5 SSBl and SSB2; SSAl5 SSA2, SSA45 SSE2, SSBl and SSB2; SSAl5 SSA2, SSEl5 SSE2, SSBl and SSB2; SSAl5 SSA3, SSA4, SSEl, SSE2 and SSBl; SSAl5 SSA3, SSA4, SSEl5 SSE2 and SSB2; SSAl5 SSA3, SSA4, SSEl5 SSBl and SSB2; SSAl, SSA3, SSA4, SSE2, SSBl and SSB2; SSAl, SSA3, SSEl5 SSE2, SSBl and SSB2; SSAl, SSA4, SSEl5 SSE2, SSBl and SSB2; SSA2, SSA3, SSA4, SSEl5 SSE2 and SSBl; SSA2, SSA3, SSA4, SSEl5 SSE2 and SSB2; SSA2, SSA3, SSA4, SSEl, SSBl and SSB2; SSA2, SSA3, SSA4, SSE25 SSBl and SSB2; SSA2, SSA3, SSEl5 SSE2, SSBl and SSB2; SSA25 SSA4, SSEl5 SSE2, SSBl and SSB2; or SSA3, SSA4, SSEl5 SSE2, SSBl and SSB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of seven of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. For example, one of the following combinations may or may not be chosen —
SSAl, SSA2, SSA3, SSA4, SSEl5 SSE2 and SSBl; SSAl5 SSA2, SSA3, SSA4, SSEl5 SSE2 and SSB; SSAl, SSA2, SSA3, SSA4, SSEl5 SSBl and SSB2; SSAl5
SSA2, SSA3, SSA4, SSE2, SSBl and SSB2; SSAl5 SSA2, SSA3, SSEl5 SSE2, SSBl and SSB2; SSAl5 SSA2, SSA4, SSEl3 SSE2, SSBl and SSB2; SSAl, SSA3, SSA4, SSEl5 SSE2, SSBl and SSB2; or SSA2, SSA3, SSA4, SSEl, SSE2, SSBl and SSB2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of all eight of the above chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation. In other words, the following combination may or may not be chosen -.
SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl and SSB2.
Mitochondrial chaperone and translocation proteins
Mitochondrial chaperone and translocation proteins include ECMlO5 MDJl, MDJ2. A detailed description of these proteins and their genes is given separately below.
In one embodiment, the host cell may or may not be genetically modified to cause over-expression of one of the above mitochondrial chaperone and translocation proteins.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of two of the above mitochondrial chaperone and translocation proteins. For example, one of the following combinations may or may not be chosen -
ECMlO and MDJl; ECMlO and MDJ2; or MDJl and MDJ2.
In another embodiment, the host cell may or may not be genetically modified to cause over-expression of all three of the above mitochondrial chaperone and translocation proteins. In that case the following combination may or may not be chosen — 02289
ECMIO5 MDJl and MDJ2.
Other Combinations ofChaperones
The skilled person will appreciate that it is possible to combine genes that encode one or more proteins from the above-defined groups of chaperones.
Thus, the host cell may or may not be genetically modified to cause simultaneous over-expression of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, "fourteen, fifteen, sixteen or seventeen of the chaperones selected from the group consisting of JEMl, LHSl, SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl and MDJ2.
Where the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined chaperones, it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins and the one or two other helper proteins may or may not be helper proteins involved in disulphide bond formation or protein degradation, as discussed below.
Over-expression of one (or more) of the ER luminal localised chaperones may or may not be combined with the over-expression of at least one of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation and/or the over-expression of at least one of the mitochondrial chaperone and translocation proteins.
For example, any one of the following combinations may or may not be chosen -
• SCJl in combination with any one, two, three, four, five, six, seven, eight, nine, ten or eleven of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl,
SSB2, ECMlO, MDJl or MDJ2; or • FKB2 in combination with any one, two, three, four, five, six, seven, eight, nine, ten or eleven of SSAl5 SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl5 SSB2, ECMlO5 MDJl or MDJ2.
• JEMl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SS A2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
• LHSl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
• SCJl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
• KAR2 in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SS A3, SS A4, SSEl5 SSE2, SSBl, SSB2 and/or in combination with ECMlO, MDJl and MDJ2;
• SILl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO5 MDJl and MDJ2; or
• FKB2 in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SSA2, SS A3, SS A4, SSEl, SSE2, SSBl, SSB2 and/or in combination with ECMlO5 MDJl and MDJ2.
Alternatively, for example, one (or more) of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation may or may not be simultaneously over-expressed with at least one of the ER luminal localised chaperones and/or at least one of the mitochondrial chaperone and translocation proteins. For example, the following combinations may or may not be chosen -
• SSEl in combination with any one, two, three, four, five, six, seven, eight or nine of JEMl, LHSl, SCJl, KAR2, SILl, FKB2, ECMlO, MDJl or MDJ2.
• SSAl in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMl 0, MDJl and MD J2;
• SSA2 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMlO, MDJl and MDJ2;
• S S A3 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMl 0, MDJl and MD J2;
• SSA4 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMl 0, MDJl and MD J2; ■ • SSEl in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl5 LHSl5 SCJl5 KAR2, SILl and FKB2 and/or in combination with ECMlO5 MDJl and MDJ2;
• SSE2 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl5 LHSl, SCJl5 KAR2, SILl and FKB2 and/or in combination with ECMlO5 MDJl and MDJ2;
• SSBl in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl5 LHSl5 SCJl5 KAR2, SILl and FKB2 and/or in combination with ECMlO5 MDJl and MDJ2; or
• SSB2 in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and/or in combination with ECMl O5 MDJl and MD J2.
Alternatively, one of the mitochondrial chaperone and translocation proteins may or may not be simultaneously over-expressed with at least one of the chaperones involved in cytoplasmic folding and maintenance of proteins in a translocation competent state prior to translocation and/or at least one of the ER luminal localised chaperones.
For example, one of the following combinations may or may not be chosen —
• ECMlO in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SJXl and FKB2;
• ECMlO in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4,
SSEl, SSE2, SSBl, SSB2; • ECMlO in combination with any of the above-listed combinations of one, two, three of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• ECMlO in combination with any of the above-listed combinations of four of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• ECMlO in combination with any of the above-listed combinations of five of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• ECMlO in combination with all six of JEMl, LHSl, SCJl, KAR2, SELl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• MDJl in combination with any of the above-listed combinations of one, two, three, four, five or six of JEMl, LHSl, SCJl, KAR2, SILl and FKB2;
• MDJl in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• MDJl in combination with any of the above-listed combinations of one, two, three of JEMl5 LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2; • MDJl in combination with any of the above-listed combinations of four of JEMl5 LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl, SSB2;
• MDJl in combination with any of the above-listed combinations of five of JEMl5 LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl, SSB2;
• MDJl in combination with all six of JEMl , LHS 15 SCJl , KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2;
• MDJ2 in combination with any of the above-listed combinations of one, two5 three, four, five or six of JEMl, LHSl5 SCJl5 KAR2, SILl and FKB2;
• MDJ2 in combination with any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• MDJ2 in combination with any of the above-listed combinations of one, two, three of JEMl, LHSl5 SCJl5 KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl5 SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl5 SSB2;
• MD J2 in combination with any of the above-listed combinations of four of JEMl, LHSl, SCJl5 KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2;
• MDJ2 in combination with any of the above-listed combinations of five of JEMl, LHSl, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2; or
• MDJ2 in combination with all six of JEMl , LHS 1, SCJl, KAR2, SILl and FKB2 and any of the above-listed combinations of one, two, three, four, five, six, seven or eight of SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2.
In another embodiment, representative members of each of the above three groups of chaperone proteins (such as one member of each group) may or may nqt be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
JEMl, SSAl and ERMlO; JEMl, SSAl and MDJl; JEMl, SSAl and MDJ2; JEMl, SSA2 and ERMlO; JEMl, SSA2 and MDJl; JEMl, SSA2 and MDJ2;
JEMl, SSA3 and ERMlO; JEMl, SSA3 and MDJl; JEMl, SSA3 and MDJ2;
JEMl, SSA4 and ERMlO; JEMl, SS A4 and MDJl; JEMl, SSA4 and MDJ2;
JEMl, SSEl and ERMlO; JEMl, SSEl and MDJl; JEMl, SSEl and MDJ2;
JEMl, SSE2 and ERMlO; JEMl, SSE2 and MDJl; JEMl, SSE2 and MDJ2; JEMl, SSBl and ERMlO; JEMl, SSBl and MDJl; JEMl, SSBl and MDJ2;
JEMl, SSB2 and ERMlO; JEMl, SSB2 and MDJl; JEMl, SSB2 and MDJ2;
LHSl,- SSAl and ERMlO; -.LHSl, SSAl and MDJl; LHSl5 SSAl and MDJ2;
LHSl, SSA2 and ERMlO; LHSl, SSA2 and MDJl; LHSl, SSA2 and- MDJ2;
LHSl, SSA3 and ERMlO; LHSl, SSA3 and MDJl LHSl, SSA3 and. MDJ2; LHSl, SSA4 and ERMlO; LHSl, SSA4 and MDJl; LHSl, SSA4 and;MDJ2;
LHSl, SSEl and ERMlO; LHSl, SSEl and MDJl; LHSl, SSEl and MDJ2;
LHSl3 SSE2 and ERMlO; LHSl, SSE2 and MDJl; LHSl, SSE2 and MDJ2; LHSl, SSBl and ERMlO; LHSl, SSBl and MDJl; LHSl, SSBl and MDJ2;
LHSl, SSB2 and ERMlO; LHSl5 SSB2 and MDJl; LHSl5 SSB2 and MDJ2;
SCJl5 SSAl and ERMlO; SCJl5 SSAl and MDJl; SCJl5 SSAl and MDJ2;
SCJl5 SSA2 and ERMlO; SCJl5 SSA2 and MDJl; SCJl, SSA2 and MDJ2; SCJl, SSA3 and ERMlO; SCJl, SSA3 and MDJl; SCJl, SSA3 and MDJ2;
SCJl5 SSA4 and ERMlO; SCJl, SSA4 and MDJl; SCJl, SSA4 and MDJ2;
SCJl5 SSEl and ERMlO; SCJl5 SSEl and MDJl; SCJl5 SSEl and MDJ2;
SCJl5 SSE2 and ERMlO; SCJl5 SSE2 and MDJl; SCJl5 SSE2 and MDJ2;
SCJl5 SSBl and ERMlO; SCJl, SSBl and MDJl; SCJl5 SSBl and MDJ2; SCJl, SSB2 and ERMlO; SCJl5 SSB2 and MDJl; SCJl5 SSB2 and MDJ2;
KAR2, SSAl and ERMlO; KAR2, SSAl and MDJl; KAR2, SSAl and MDJ2;
KAR2, SSA2 and ERMlO; KAR2, SSA2 and MDJl; KAR2, SS A2 and MDJ2;
KAR2, SS A3 and ERMlO; KAR2, SS A3 and MDJl; KAR2, SS A3 and MDJ2;
KAR25 SS A4 and ERMlO; KAR25 SSA4 and MDJl; KAR2, SSA4 and MDJ2; KAR2, SSEl and ERMlO; KAR2, SSEl and MDJl; KAR2, SSEl and MDJ2;
KAR2, SSE2 and ERMlO; KAR2, SSE2 and MDJl; KAR2, SSE2 and MDJ2;
KAR2, SSBl and ERMlO; KAR25 SSBl and MDJl; KAR2, SSBl and MDJ2;
KAR2, SSB2 and ERMlO; KAR2, SSB2 and MDJl; KAR2, SSB2 and MDJ2;
SJXl5 SSAl and ERMlO; SILl5 SSAl and MDJl; SILl5 SSAl and MDJ2; SILl, SSA2 and ERMlO; SILl5 SSA2 and MDJl; SILl5 SSA2 and MDJ2; SILl5
SSA3 and ERMlO; SILl, SSA3 and MDJl; SILl5 SSA3 and MDJ2; SILl5
SSA4 and ERMlO; SILl, SS A4 and MDJl; SILl5 SSA4 and MDJ2; SILl5 SSEl and ERMlO; SILl, SSEl and MDJl; SILl, SSEl and MDJ2; SILl5 SSE2 and
ERMlO; SILl, SSE2 and MDJl; SILl5 SSE2 and MDJ2; SILl5 SSBl and ERMlO; SILl, SSBl and MDJl; SILl5 SSBl and MDJ2; SILl5 SSB2 and
ERMlO; SILl, SSB2 and MDJl; SILl5 SSB2 and MDJ2; FKB2, SSAl and
ERMlO; FKB2, SSAl and MDJl; FKB2, SSAl and MDJ2; FKB2, SSA2 and
ERMlO; FKB25 SSA2 and MDJl; FKB2, SSA2 and MDJ2; FKB2, SS A3 and
ERMlO; FKB2, SSA3 and MDJl; FKB2, SSA3 and MDJ2; FKB2, SSA4 and ERMlO; FKB2, SSA4 and MDJl; FKB2, SSA4 and MDJ2; FKB2, SSEl and .
ERMlO; FKB2, SSEl and MDJl; FKB2, SSEl and MDJ2; FKB2, SSE2 and
ERMlO; FKB2, SSE2 and MDJl; FKB2, SSE2 and MDJ2; FKB2, SSBl and ERMlO; FKB2, SSBl and MDJl; FKB2, SSBl and MDJ2; FKB2, SSB2 and ERMlO; FKB2, SSB2 and MDJl; or FKB2, SSB2 and MDJ2.
The skilled person will also appreciate that any of the above defined combinations may or may not also be combined with any of the following genes or combinations of genes encoding other helper proteins, in particular helper proteins involved in disulphide bond formation or helper proteins involved in protein degradation, as discussed below.
Proteins involved in disulphide bond formation
Proteins involved hi the formation of disulphide bonds in other proteins include EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl. A detailed description of these proteins and their genes is given separately below.
In one embodiment, one of the above disulphide bond formation proteins may or may not be over-expressed in the host cell. For example, ERV2 may or may not be chosen.
In another embodiment, two of the above disulphide bond formation proteins may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
• EROl in combination with one of ERV2, EUGl, MPDl, MPD2, EPSl or PDIl;
• ERV2 in combination with one of EUGl , MPD 1 , MPD2, EPS 1 or PDIl ;
• EUGl in combination with one of MPDl, MPD2, EPSl or PDIl;
• MPDl in combination with one of MPD2, EPSl or PDIl;
• MPD2 in combination with one of EPS 1 or PDIl ; or ■ ' • EPSl in combination with PDIl. In another embodiment, three of the above helper proteins may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen —
EROl, ERV2 and EUGl; EROl, ERV2 and MPDl; EROl, ERV2 and MPD2;
EROl, ERV2 and EPSl; EROl, ERV2 and PDIl; EROl, EUGl and MPDl;
EROl, EUGl and MPD2; EROl, EUGl and EPSl; EROl, EUGl and PDIl;
EROl5 MPDl and MPD2; EROl, MPDl and EPSl; EROl, MPDl and PDIl;
EROl, MPD2 and EPSl; EROl, MPD2 and PDIl; EROl, EPSl and PDIl; ERV2, EUGl and MPDl; ERV2, EUGl and MPD2; ERV2, EUGl and EPSl;
ERV2, EUGl and PDIl; ERV2, MPDl and MPD2; ERV2, MPDl and EPSl;
ERV2, MPDl and PDIl; ERV2, MPD2 and EPSl; ERV2, MPD2 and PDIl;
ERV2, EPSl and PDIl; EUGl5 MPDl and MPD2; EUGl, MPDl and EPSl;
EUGl5 MPDl and PDIl; EUGl, MPD2 and EPSl; EUGl, MPD2 and PDIl; EUGl, EPSl and PDIl; MPDl5 MPD2 and EPSl; MPDl, MPD2 and PDIl;
MPDl5 EPSl and PDIl; or MPD2, EPSl and PDIl.
In another embodiment, four of the above helper proteins may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
EROl5 ERV2, EUGl and MPDl; EROl5 ERV2, EUGl and MPD2; EROl5 ERV2, EUGl and EPSl; EROl5 ERV2, EUGl and PDIl; EROl5 ERV2, MPDl and MPD2; EROl, ERV2, MPDl and EPSl; EROl5 ERV2, MPDl and PDIl; EROl5 ERV2, MPD2 and EPSl; EROl5 ERV2, MPD2 and PDIl; EROl5 ERV2, EPSl ■ and PDIl; EROl, EUGl5 MPDl and MPD2; EROl5 EUGl, MPDl and EPSl; EROl5 EUGl, MPDl and PDIl; EROl5 EUGl5 MPD2 and EPSl; EROl5 EUGl, MPD2 and PDIl; EROl, EUGl5 EPSl and PDIl; EROl5 MPDl5 MPD2 and EPSl; EROl, MPDl, MPD2 and PDIl; EROl5 MPDl5 EPSl and PDIl; EROl5 MPD25 EPSl and PDIl; ERV2, EUGl5 MPDl and MPD2; ERV2, EUGl, MPDl and EPSl; ERV2, EUGl, MPDl- and PDIl; ERV2, EUGl5 MPD2 and EPSl; ERV2, EUGl5 MPD2 and PDIl; ERV2, EUGl5 EPSl and PDIl; ERV2, MPDl5
MPD2 and EPSl; ERV2, MPDl5 MPD2 and PDIl; ERV2, MPDl5 EPSl and PDIl; ERV2, MPD2, EPSl and PDIl; EUGl, MPDl5 MPD2 and EPSl; EUGl5 MPDl5 MPD2 and PDIl; EUGl5 MPDl5 EPSl and PDIl; EUGl5 MPD2, EPSl and PDIl; or MPDl5 MPD2, EPSl and PDIl.
In another embodiment, five of the above helper proteins may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
EROl5 ERV2, EUGl5 MPDl and MPD2; EROl5 ERV2, EUGl5 MPDl and EPSl; EROl5 ERV2, EUGl, MPDl and PDIl; EROl5 ERV2, EUGl5 MPD2 and EPSl;
EROl5 ERV2, EUGl5 MPD2 and PDIl; EROl5 ERV2, EUGl5 EPSl and PDIl;
EROl5 ERV25 MPDl, MPD2 and EPSl; EROl5 ERV2, MPDl, MPD2 and PDIl;
EROl5 ERV2, MPDl, EPSl and PDIl; EROl, ERV2, MPD2, EPSl and PDIl;
EROl, EUGl, MPDl, MPD2 and EPSl; EROl, EUGl, MPDl5 MPD2 and PDIl; EROl5 EUGl5 MPDl5 EPSl and PDIl; EROl5 EUGl, MPD2, EPSl and PDIl;
EROl5 MPDl5 MPD2, EPSl and PDIl; ERV2, EUGl5 MPDl5 MPD2 and EPSl;
ERV2, EUGl, MPDl5 MPD2 and PDIl; ERV25 EUGl5 MPDl, EPSl and PDIl;
ERV2, EUGl5 MPD2, EPSl and PDIl; ERV2, MPDl5 MPD25 EPSl and PDIl; or
EUGl5 MPDl5 MPD2, EPSl and PDIl
In another embodiment, six of the above helper proteins may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
EROl5 ERV25 EUGl5 MPDl5 MPD2 and EPSl; EROl5 ERV2, EUGl5 MPDl5 MPD2 and PDIl; EROl, ERV2, EUGl5 MPDl5 EPSl and PDIl; EROl5 ERV2, EUGl5 MPD2, EPSl and PDIl; EROl5 ERV2, MPDl5 MPD25 EPSl and PDIl; EROl5 EUGl5 MPDl5 MPD2, EPSl and PDIl; or ERV2, EUGl, MPDl5 MPD2, EPSl and PDIl.
It is anticipated that EROl and ERV2 may function independently of each other or they may co-operate. Therefore, in one embodiment disclosure of EROl may or may not also include the combinations of EROl and ERV2, or ERV2 in its place. Similarly, in another embodiment disclosure of ERV2 may or may not also include the combinations of ERV2 and EROl, or EROl in its place.
In another embodiment, all seven of the above helper proteins may or may not be simultaneously over-expressed in the host cell. In that case, the following combinations may or may not be chosen -
EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
Where the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined disulphide bond formation helper proteins, it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins and the one or two other helper proteins may or may not be chaperones or helper proteins involved in protein degradation, as discussed above, and below, respectively.
Where one of the helper proteins is a protein disulphide isomerase, such as a yeast and mammalian PDI, mammalian Erp59, mammalian prolyl-4-hydroxylase B- subunit, yeast GSBP, yeast EUGl and mammalian T3BP, then it may or may not be preferred, in one embodiment, to avoid co-expression with KAR2 or an equivalent thereof including hsp chaperone proteins such as other yeast Hsp70 proteins. BiP, SSAl -4. SSBL SSCl and SSDl gene products and eukaryotic hsp70 proteins such as HSP68, HSP72, HSP73, HSC70, clathrin uncoating ATPase, IgG heavy chain binding protein (BiP)3 glucose-regulated proteins 75, 78 and 80 (GRP75, GPR78 and GRP80) and the like, particularly where these are the sole helper proteins that are overexpressed in the host cell.
Proteins involved in protein degradation
Proteins involved in protein degradation include DERI, DER3, HRD3, UJBC7 and D0A4. A detailed description of these proteins and their genes is given separately below. In one embodiment, one of the above proteins involved in protein degradation may or may not be over-expressed in the host cell. For example, DERI may or may not be chosen, DER3 may or may not be chosen, HRD3 may or may not be chosen, UBC7 may or may not be chosen, or D0A4 may or may not be chosen.
In another embodiment, two of the above proteins involved m protein degradation may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; HRD3 and UBC7; HRD3 and D0A4; or UBC7 and D0A4.
In another embodiment, three of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
DERI, DER3 and HRD3; DERI, DER3 and UBC7; DERI, DER3 and D0A4; DERI, HRD3 and UBC7; DERI, HRD3 and DOA4; DERI, UBC7 and DOA4; DER3, HRD3 and UBC7; DER3, HRD3 and DOA4; DER3, UBC7 and DOA4; or HRD3, UBC7 and DOA4.
In another embodiment, four of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell. For example, one of the following combinations may or may not be chosen -
DERI, DER3, HRD3 and UBC7; DERI, DER3, HRD3 and D0A4; DERI, DER3, UBC7 and DOA4; DERI, HRD3, UBC7 and D0A4; or DER3, HRD3, UBC7 and DOA4.
In another embodiment, all five of the above proteins involved in protein degradation may or may not be simultaneously over-expressed in the host cell. In that case, the following combination is chosen - DERl5 DER3, HRD3, UBC7 andDOA4.
Where the host cell is genetically modified to cause simultaneous over-expression of one or two of the above defined protein degradation helper proteins, it may or may not be preferred that the host cell is genetically modified to cause simultaneous over-expression of at least three helper proteins in total and the one or two other helper proteins may or may not be chaperones or disulphide bond formation helper proteins, as discussed above.
HACl (encoded by a spliced or unspliced polynucleotide)
Valkonen et al. 2003 {Applied Environ. Micro., 69, 2065) reported investigations into the possibility to obtain better yields of secreted proteins. The authors found that the manipulation of the unfolded-protein response (UPR) pathway regulator, HACl, affected the production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. For example, it is reported that constitutive over- expression of HACl caused a 70% increase in alpha-amylase secretion. WO 01/72783 also reports that. HACl overexpression can be used to increase the secretion of a heterologous protein in a eukaryotic cell by inducing an elevated UPR5 and PTC2 and IREl are also suggested for use in place of HACl.
Over-expression of HACl can be achieved, for example, by the introduction of a recombinant polynucleotide that comprises the endogenous HACl gene coding sequence or a truncated intronless HACl coding sequence (Valkonen et al. 2003, Applied Environ. Micro., 69, 2065). A detailed description of this protein and its gene is given separately below. The same techniques can be used to over-express PTC2 or IREl.
In one embodiment of the present invention, a host cell of the present invention may or may not be genetically engineered to cause over-expression HACl, PTC2 or IREl , such as by modification of an endogenous gene encoding HACl, PTC2 or IREl, or by transformation with a recombinant gene encoding HACl5 PTC2 or IREl. For example HACl, PTC2 or IREl may or may not be simultaneously over-expressed with any of the above-defined combinations of other helper proteins.
In one embodiment, the host cell of the present invention is not genetically engineered to cause HACl over-expression, such as by modification of an endogenous HACl gene or transformation with a recombinant HACl gene.
In another embodiment where the host cell is genetically engineered to cause over- expression of HACl, PTC2 or IREl, the host cell is additionally genetically modified by the introduction of at least one recombinant gene encoding at least one other helper protein, such as a DnaJ-like protein, an Hsp70 family protein and/or SILl or by the modification of the sequence of an endogenous gene encoding one or more other helper proteins at least one of a DnaJ-like protein, an Hsp70 family protein (such as LHSl) and SILl to cause increased expression of the thus modified gene.
Other combinations
In light of the above disclosure, the skilled person will appreciate that the present invention also encompasses simultaneous over-expression of any combination of helper proteins derived from any of the above-defined groups.
For example, two . helper proteins may or may not be simultaneously over- expressed. Suitable combinations include any one of the following combinations:
JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and
FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4;
JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and
ECMl 0; JEMl and MDJl ; JEMl and MDJ2; JEMl and EROl ; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl;. JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and
UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2;
LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPS 1 ; SCJl and PDIl ; SCJl and DERI ; SCJl and DER3 ; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI;
SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SS A3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and
ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2;
" SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and' PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl ; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; EGMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and
MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ER.V2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl ; MDJ2 and EROl ; MDJ2 and ERV2; MDJ2 and EUGl ; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; DOA4 and HACl.
The skilled person will also appreciate that the present invention encompasses simultaneous over-expression of at least three helper proteins, and that the at least three helper proteins may or may not be taken from any combination of helper proteins derived from any of the above-defined groups. For example, one of the following combinations of three helper proteins may or may not be simultaneously over-expressed, with or without the over-expression of one or more additional helper proteins:
JEMl in combination with any one of the following combinations: LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3;- KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; ..
SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and
■ EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and D0A4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and
MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FICB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SS A2; SSAl and SS A3; SSAl and SS A4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and 5 EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SS A2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 0 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 5 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and
DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4;
SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and
SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; •
■ SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; . SSEl andHRD3; SSEl andUBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and
HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and D0A4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl arid DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl. LHSl in combination with any one of the following combinations: JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and. EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl. and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SS A3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and T/GB2006/002289
EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and D0A4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SS A2 and SSE2; SS A2 and SSBl; SS A2 and SSB2; SS A2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 andDOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl andUBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and
DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and 2006/002289
EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; 5. MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and 0 DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl . and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl ; or DOA4 and HACl .
SCJl in combination with any one of the following combinations: JEMl and LHSl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SS A2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and
SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; B2006/002289
JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4; JEMl and HACl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2;.SIL1 and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SS A3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SS A2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SS A3 and DER3; SS A3 and HRD3; SS A3 and UBC7; SS A3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMl 0 and MDJ2; ECMl 0 and ERO 1 ; ECMl 0 and ERV2; ECMl 0 and EUGl ; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; . ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7;
ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl ; ERV2 and MPD2; ERV2 and EPS 1 ; ERV2 and PDIl ; ERV2 and DERI ; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and D0A4; UBC7 and HACl; or D0A4 and HACl.
KAR2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and
DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl, and. ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2;- FKB2 and ECMl 0; FKB2 and MDJl ; FKB2 and MD J2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2'and MPD2; FKB2 and EPS 1 ; FKB2 and PDIl ;. FKB2 and DERI ; FKB2 and DER3 ;
FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SS A2 and DERI;
SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SS A3 and EPSl; SS A3 and PDIl; SS A3 and DERI; SS A3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl ; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and • HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and
DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPDl and HACl; MPD2 and EPS1;..MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
SILl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and FKB2;- JEMl and SSAl; JEMl and SSA2; JEMl and SS A3; JEMl and SS A4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl;
LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and FKB2; SCJl and
SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl;
SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and
HACl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and ' EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3;' SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and. HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SS A3 and MDJ2; SSA3 and EROl; SS A3 and ERV2;
SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; S5A4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SS A4 and EROl; SSA4 and ERV2; SS A4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and-ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and
ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl. and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and D0A4; UBC7 and HACl; or D0A4 and HACl.
FKB2 in combination with any one of the following combinations: JEMl and
LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and SSAl;
JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and
SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl;
JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and
DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4;
JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and
SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl;
LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and
EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl;
LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SJXl; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; ICAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SS A3 and MDJ2; SS A3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and
SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SS A4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SS A4 and MPD2; SS A4 and EPSl ; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 andUBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and - HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl;
ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ER.V2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and
MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3;
EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl;
MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and
HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3;
MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and
UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and
DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl;
DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and
HACl ; or D0A4 and HACl .
SSAl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and
SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and ,
DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4;
JEMl and HACl ; LHS 1 and SCJl ; LHS 1 and KAR2; LHS 1 and SILl ; LHS 1 and
FKB2; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and
EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl;
LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHS1 and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and
SSE2; SCJl and SSBl; SCJl and SSB2;' SCJl and ECMlO; SCJl and MDJl;
SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and
MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and - MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and .MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SS A2 and D0A4; SS A2 and HACl; SS A3 and SSA4; SS A3 and SSEl; SS A3 and SSE2; SSA3 and SSBl; SS A3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SS A3 and HRD3; SSA3 and UBC7; SSA3 and D0A4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2;.SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SS A4 and DERI; SS A4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and D0A4; SSA4 and HACl; SSEl and SSE2; SSEl and
SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and D0A4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; 5 SSE2 and EROl ; SSE2 and ERV2; SSE2 and EUGl ; SSE2 and MPD 1 ; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2;
10 SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and
15 DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl;
20 MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; .MD J2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and
25 DOA4; MDJ2 and HACl ; EROl and ERV2; EROl and EUGl ; EROl and MPDl ;
. EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and
' HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2
30 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl;
MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
SSA2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and ' SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHS 1 and HACl ; SCJl and KAR2; SCJl and SILl ; SCJl and FKB2; SCJl and SSAl; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and. HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and . PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl andDOA4; SSAl and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SS A4 and PDIl; SS A4 and DERI; SS A4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl;' SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl andHRD3; SSEl andUBC7; SSEl andDOA4; SSEl and HACl; SSE2 and
SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl ; ERV2 and DERI ; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; ■ EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
SSA3 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SJJLl; SCJl and FKB2;
SCJl and SSAl; SCJl and SSA2; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl;
SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; . FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl andHRD3; SSEl andUBC7; SSEl andDOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl ; SSE2 and ERV2; SSE2 and EUGl ; SSE2 and MPDl ; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl ; ERV2 and EUGl ; ERV2 and MPD 1 ; ERV2 and MPD2; ERV2 and EPS 1 ; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl;. MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl ; MPD2 and EPS 1 ; MPD2 and PDIl ; MPD2 and DERI ; MPD2 and DER3 ; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl;
DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or DOA4 and HACl.
SSA4 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl . and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl ; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPS 1 ; KAR2 and PDIl ; KAR2 and DERI ; KAR2 and DER3; . ' KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI;
SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FICB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SS A3; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl ; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl andUBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO;
SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HAC 1 ; MDJ2 and ERO 1 ; MD J2 and ERV2; MD J2 and EUGl ; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl;. EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl. SSEl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SJXl and EUGl; SELl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SDLl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and 02289
EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SS A2; SSAl and SSA3; SSAl and SSA4; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and
MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SS A4 and EPS 1 ; SS A4 and PDIl ; SS A4 and DERI ; SS A4 and
DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl;
SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and
MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl;
SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl;
SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and
MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3;
SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and -EROl; SSB2 and
ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl;
SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and
UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl ;• UBC7 and DOA4; UBC7 and HACl ; or DOA4 and HACl .
SSE2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and
SSEl; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SS A3; -LHSl and SSA4; LHSl and SSEl; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPS 1 ; KAR2 and PDIl ; KAR2 and DERI ; KAR2 and DER3 ; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SJXl and SSA4; SILl and SSEl; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and
SSA4; SSAl and SSEl; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl;. SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl;
SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and
UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and
DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl ; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and . HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
SSBl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and . SSEl; JEMl and SSE2; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4;
JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7;
SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and
MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl;
SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and
DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and
SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl;
SSA3 and ERV2; SSA3 and EUGl; S S A3 and MPDl; SSA3 and MPD2; SSA3
' and EPS 1 ; SSA3 and PDIl ; SSA3 and DERI ; SSA3 and DER3 ; SSA3 and HRD3 ;
SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and
HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSB2 and ECMlO;
SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and
EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl;
. SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl;
MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl ; ERV2 and EUGl ; ERV2 and MPD 1 ; ERV2 and MPD2; ERV2 and EPS 1 ; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPD 1 and MPD2; MPD 1 and EPS 1 ; MPD 1 and PDIl ; MPD 1 and DERI ; MPD 1 and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl ; or D0A4 and HACl .
SSB2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and
EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and
DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SS A3 and ECMlO; SS A3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl andDER3; SSBl andHRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl ; MDJl and MDJ2; MDJl and EROl ; MDJl and ERV2; MDJl and EUGl ; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2. and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl ; or D0A4 and HACl .
ECMlO in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJLl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SS A2; LHSl and SSA3; LHSl and SS A4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl. and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and D0A4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and
SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; ICAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SS A3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl ■ and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SS A2; FKB2 and SS A3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FICB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SS A3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4-; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl;. MDJ2 and EROl; . MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPD 1 ; ERV2 and MPD2; ERV2 and EPS 1 ; ERV2 and PDIl ; ERV2 and DERI ; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and
PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERl; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
MDJl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl 'and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SS A3; LHSl and SS A4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMl 0; LHS 1 and MD J2; LHS 1 and ERO 1 ; LHS 1 and ERV2; LHS 1 and EUGl ; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl;
KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SS A3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl ; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4;
SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJ2; SSA3 and EROl;
SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4;. SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl;
SSA4 andMPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and
91 DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4;
MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
MDJ2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and
MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and
DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4;
SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and EROl;
SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl;
SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl;
SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and
EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2;
SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2;
SSBl and ECMlO; SSBl and MDJl; SSBl and EROl; SSBl and ERV2; SSBl ■ and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and
PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7;
SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and
MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3;
SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and EROl; MDJl and ERV2; MDJl and
EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl;
MDJl and DERI; MDJl and DER3; MDJl and HRD3; KdDJl and UBC7; MDJl and DOA4; MDJl and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI;
EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and
EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3;
ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and
DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and
HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI;
MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4;
MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and.
HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3;
EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and
DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
EROl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and
ECMlO; SILl and MDJl; SILl and MDJ2; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMl 0; FKB2 and MDJl ; FKB2 and MDJ2; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and ERV2; SSA2 and EUGl; SS A2 and MPDl; SS A2 and MPD2; SS A2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2;
SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and D0A4; SSE2 and HACl; SSBl and SSB2;
SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and
PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and
MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3;
SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and ERV2; MDJl and
EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl;
MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and
MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI;
MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2. and
EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl ; EUGl and MPDl ; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and
DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and
HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI;
MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and
HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3;
EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and
DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and
HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and
DOA4; UBC7 and HACl; or D0A4 and HACl. ERV2 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJXl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl;' LHSl and MDJ2; LHSl and EROl; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPS 1 ; KAR2 and PDIl ; KAR2 and DERI ; KAR2 and DER3 ; KAR2 and HRD3; ICAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3;. SIL1 and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and EUGl; SHl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SJLl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and
SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl. and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SS A2 and DER3; SS A2 and HRD3; SS A2 and UBC7; SS A2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2' and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and
PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and • DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl;- HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HAC 1 ; or D0A4 and HAC 1.
EUGl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4;' SILl and HACl; FKB2 and SSAl; FKJB2 and.SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMl 0; FKB2 and MDJl ; FKB2 and MD J2; FKB2 and ERO 1 ; FKB2 and ERV2; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7;
FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and MPDl; SSA2 and MPD2; SS A2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and MPDl; SS A4 and MPD2; SS A4 and EPSl; SS A4 and PDIl; SSA4 and DERI; SS A4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl andHRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3;
SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and MPDl;
ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI;
ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4;
ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and
DOA4; MDJl and HACl; MD J2 and EROl; MDJ2 and ERV2; MDJ2 and MPDl;
MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and MPDl; EROl and MPD2; EROl and EPSl;
EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and MPDl; ERV2 and
MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3;
ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and
DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and
HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3;
MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl;
EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3;
PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and
DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; .DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
MPDl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SJXl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl;. JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and
DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; 2006/002289
JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl andHRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and
DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; 89
SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SS A2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SS A3 and EUGl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl andMDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl ; SSE2 and MPD2; SSE2 and EPSl ; SSE2 and PDIl ; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and
MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl ; EUGl and MPD2; EUGl and EPSl ; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and D0A4; UBC7 and HACl; or D0A4 and HACl.
MPD2 in combination with any one of "the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMI and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4; JEMl and HACl ; LHS 1 and SCJl ; LHS 1 and KAR2; LHS 1 and SILl ; LHS 1 and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and
ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 andDER3; SSB2 and HRD3; SSB2 andUBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and EPSl;
MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; ERV2 and HACl; EUGl and MPDl; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; EUGl and HACl; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPDl and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and D0A4; DER3 and HACl; HRD3 and UBC7; HRD3 and D0A4; HRD3 and HACl; UBC7 and D0A4; UBC7 and HACl; or D0A4 and HACl.
EPSl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and
SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO;
JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and D0A4;
JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and
FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4;
LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and
ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and
D0A4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2;
SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; ICAR2 and SSB2; KAR2 and ECMlO; ICAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and F5CB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2. and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SS A4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and PDIl; SSA4 and DER1;' SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and • EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO antf DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl' and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 .and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and PDIl; EROl and DERI;
EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl ; EUGl and MPDl ; EUGl and MPD2; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or DOA4 and HACl.
PDIl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl .and FKB2; SCJl and SSAl; SCJl and SS A2; SCJl and SSA3; SCJl and SS A4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl;
SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and DERI; SCJl and DER3; SCTl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SS A3; KAR2 and SS A4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl ; FKB2 and MDJ2; FKB2 and EROl ; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMl 0; SSA2 and MDJl ; SSA2 and MD J2; SSA2 and EROl ; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SS A3 and SSBl; SS A3 and SSB2; SS A3 and ECMlO; SS A3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3- and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and DERI; SSE2 and DER3; SSE2 and IΪRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and ERO 1 ; ECMl 0 and ERV2; ECMl 0 and EUGl ; ECMl 0 and MPD 1 ; ECMl 0 and MPD2; ECMlO and EPSl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and ERO 1 ; MD J2 and ERV2; MD J2 and EUGl ; MD J2 and MPD 1 ; MD J2 and MPD2; MDJ2 and EPSl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl . and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7;
ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; DERI and DER3r DERl and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or DOA4 and HACl.
DERI in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and DERI; SCJl and
DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; ICAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SDLl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4. and ECMlO; SS A4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SS A4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; 5 SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl 0 and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; 5 SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and 0 MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJi and MPDl ;
MDJl and MPD2; MDJl and EPSl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and
■ ERO 1 ; MD J2 and ERV2; MDJ2 and EUGl ; MDJ2 and MPD 1 ; MD J2 and MPD2;
MDJ2 and EPSl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 5 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; 0 ERV2 and DOA4; ERV2 and HACl ; EUGl and MPDl ; EUGl and MPD2; EUGl and EPSl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and
■ EPSl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl ; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl.
DER3 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKJ32; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl" and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SS A3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and
MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and HRD3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SS A3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl' and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SS A2 and HRD3; SS A2 and UBC7; SS A2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; 'SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SS A4 and DERI; SSA4 and HRD3; SS A4 and UBC7; SS A4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERl; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and HRD3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and ERO 1 ; MDJ2 and ERV2; MDJ2 and EUGl ; MD J2 and MPD 1 ; MD J2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and HRD3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and HRD3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and HRD3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and HRD3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and HRD3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and
HACl; EPSl and PDIl; EPSl and DERI; EPSl and HRD3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and HRD3; PDIl and UBC7; PDIl and DOA4; PDIl and HACl; DERI and HRD3; DERI and UBC7; DERI and DOA4; DERI and HACl; HRD3 and UBC7; HRD3 and DOA4; HRD3 and HACl; UBC7 and DOA4; UBC7 and HACl; or D0A4 and HACl .
HRD3 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; -JEM1 and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and UBC7; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and UBC7; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and UBC7; SCJl and DOA4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and • MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and UBC7; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SELl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and
ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and UBC7; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FICB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMl 0; FKB2 and MDJl ; FKB2 and MD J2; FKB2 and ERO 1 ; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and UBC7; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and UBC7; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and UBC7; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and UBC7; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4, and ERV2; SSA4 and EUGl; SS A4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and UBC7; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3;; SSEl and UBC7; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2;
SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3;; SSE2 and UBC7; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and UBC7; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and UBC7; SSB2 and DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and UBC7; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and UBC7; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and UBC7; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and UBC7; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and UBC7; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and UBC7; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and UBC7; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and UBC7; MPD2 and DOA4; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and UBC7; EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and . UBC7; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and UBC7; DERI and DOA4; DERI and HACl; DER3 and UBC7; DER3 and DOA4; DER3 and HACl; UBC7 and DOA4; UBC7 and HACl; or DOA4 and HACl. UBC7 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2; JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and DOA4; JEMl and HACl; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and DOA4; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and D0A4; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SS A4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and- ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and D0A4; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SJXl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and DOA4; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and
SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FICB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and DOA4; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and DOA4; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SS A2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and DOA4; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and DOA4; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and DOA4; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and DOA4; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DER1;-SSE2 and DER3; SSE2 and HRD3; SSE2 and DOA4; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and
MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and DOA4; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and 5 DOA4; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and DOA4; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl;
10 MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and DOA4; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and DOA4; MDJ2 and HACl; EROl and ERV2; EROl and
15 EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and DOA4; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and DOA4; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl
20 and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and DOA4; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and DOA4; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and DOA4; MPD2 and
25 HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; . EPSl and DOA4; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and DOA4; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and DOA4; DERI and HACl; DER3 and HRD3; DER3 and DOA4; DER3 and HACl ; HRD3 and DOA4; HRD3 and HACl ; or D0A4 and HACl . ■ 30
D0A4 in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SILl; JEMl and FKB2;
JEMl and SSAl; JEMl and SSA2; JEMl and SSA3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and HACl ; LHS 1 and SCJl ; LHS 1 and KAR2; LHS 1 and SILl ; LHS 1 and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and HACl; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and HACl; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SS A2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and HACl; SILl and FKB2; SILl and SSAl; SJXl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and HACl; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SS A4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMl 0;- FKB2 and MDJl ; FKB2 and MDJ2; FKB2 and ERO 1 ;. FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3;
FKB2 and UBC7; FKB2 and HACl; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and HACl; SSA2 and SSA3; SSA2 and SSA4; SSA2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SS A2 and MPD2; SS A2 and EPSl; SS A2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and HACl; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and HACl; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and HACl; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and HACl; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and HACl; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and HACl; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl;
SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and HACl; ECMlO and MDJl; ECMlO and MDJ2; ECMlO and EROl; ECMlO and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and HRD3; ECMlO and UBC7; ECMlO and HACl; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and HACl; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and HACl; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and HACl; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and HACl; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and HACl; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and HACl; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and HACl; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and HACl; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and HACl; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and HACl; DER3 and HRD3; DER3 and UBC7; DER3 and HACl; HRD3 and UBC7; HRD3 and HACl; or UBC7 and HACl.
HACl in combination with any one of the following combinations: JEMl and LHSl; JEMl and SCJl; JEMl and KAR2; JEMl and SIXl; JEMl and FKB2; JEMl and SSAl; JEMl and SS A2; JEMl and SS A3; JEMl and SSA4; JEMl and SSEl; JEMl and SSE2; JEMl and SSBl; JEMl and SSB2; JEMl and ECMlO; JEMl and MDJl; JEMl and MDJ2; JEMl and EROl; JEMl and ERV2; JEMl and EUGl; JEMl and MPDl; JEMl and MPD2; JEMl and EPSl; JEMl and
' PDIl; JEMl and DERI; JEMl and DER3; JEMl and HRD3; JEMl and UBC7; JEMl and DOA4; LHSl and SCJl; LHSl and KAR2; LHSl and SILl; LHSl and FKB2; LHSl and SSAl; LHSl and SSA2; LHSl and SSA3; LHSl and SSA4; LHSl and SSEl; LHSl and SSE2; LHSl and SSBl; LHSl and SSB2; LHSl and ECMlO; LHSl and MDJl; LHSl and MDJ2; LHSl and EROl; LHSl and ERV2; LHSl and EUGl; LHSl and MPDl; LHSl and MPD2; LHSl and EPSl; LHSl and PDIl; LHSl and DERI; LHSl and DER3; LHSl and HRD3; LHSl and UBC7; LHSl and DOA4; SCJl and KAR2; SCJl and SILl; SCJl and FKB2; SCJl and SSAl; SCJl and SSA2; SCJl and SSA3; SCJl and SSA4; SCJl and SSEl; SCJl and SSE2; SCJl and SSBl; SCJl and SSB2; SCJl and ECMlO; SCJl and MDJl; SCJl and MDJ2; SCJl and EROl; SCJl and ERV2; SCJl and EUGl; SCJl and MPDl; SCJl and MPD2; SCJl and EPSl; SCJl and PDIl; SCJl and DERI; SCJl and DER3; SCJl and HRD3; SCJl and UBC7; SCJl and DOA4; KAR2 and SILl; KAR2 and FKB2; KAR2 and SSAl; KAR2 and SSA2; KAR2 and SSA3; KAR2 and SSA4; KAR2 and SSEl; KAR2 and SSE2; KAR2 and SSBl; KAR2 and SSB2; KAR2 and ECMlO; KAR2 and MDJl; KAR2 and MDJ2; KAR2 and EROl; KAR2 and ERV2; KAR2 and EUGl; KAR2 and MPDl; KAR2 and MPD2; KAR2 and EPSl; KAR2 and PDIl; KAR2 and DERI; KAR2 and DER3; KAR2 and HRD3; KAR2 and UBC7; KAR2 and DOA4; SILl and FKB2; SILl and SSAl; SILl and SSA2; SILl and SSA3; SILl and SSA4; SILl and SSEl; SILl and SSE2; SILl and SSBl; SILl and SSB2; SILl and ECMlO; SILl and MDJl; SILl and MDJ2; SILl and EROl; SILl and ERV2; SILl and EUGl; SILl and MPDl; SILl and MPD2; SILl and EPSl; SILl and PDIl; SILl and DERI; SILl and DER3; SILl and HRD3; SILl and UBC7; SILl and DOA4; FKB2 and SSAl; FKB2 and SSA2; FKB2 and SSA3; FKB2 and SSA4; FKB2 and SSEl; FKB2 and SSE2; FKB2 and SSBl; FKB2 and SSB2; FKB2 and ECMlO; FKB2 and MDJl; FKB2 and MDJ2; FKB2 and EROl; FKB2 and ERV2; FKB2 and EUGl; FKB2 and MPDl; FKB2 and MPD2; FKB2 and EPSl; FKB2 and PDIl; FKB2 and DERI; FKB2 and DER3; FKB2 and HRD3; FKB2 and UBC7; FKB2 and DOA4; SSAl and SSA2; SSAl and SSA3; SSAl and SSA4; SSAl and SSEl; SSAl and SSE2; SSAl and SSBl; SSAl and SSB2; SSAl and ECMlO; SSAl and MDJl; SSAl and MDJ2; SSAl and EROl; SSAl and ERV2; SSAl and EUGl; SSAl and MPDl; SSAl and MPD2; SSAl and
EPSl; SSAl and PDIl; SSAl and DERI; SSAl and DER3; SSAl and HRD3; SSAl and UBC7; SSAl and DOA4; SSA2 and SSA3; SSA2 and SSA4; SS A2 and SSEl; SSA2 and SSE2; SSA2 and SSBl; SSA2 and SSB2; SSA2 and ECMlO; SSA2 and MDJl; SSA2 and MDJ2; SSA2 and EROl; SSA2 and ERV2; SSA2 and EUGl; SSA2 and MPDl; SSA2 and MPD2; SSA2 and EPSl; SSA2 and PDIl; SSA2 and DERI; SSA2 and DER3; SSA2 and HRD3; SSA2 and UBC7; SSA2 and DOA4; SSA3 and SSA4; SSA3 and SSEl; SSA3 and SSE2; SSA3 and SSBl; SSA3 and SSB2; SSA3 and ECMlO; SSA3 and MDJl; SSA3 and MDJ2; SSA3 and EROl; SSA3 and ERV2; SSA3 and EUGl; SSA3 and MPDl; SSA3 and MPD2; SSA3 and EPSl; SSA3 and PDIl; SSA3 and DERI; SSA3 and DER3; SSA3 and HRD3; SSA3 and UBC7; SSA3 and DOA4; SSA4 and SSEl; SSA4 and SSE2; SSA4 and SSBl; SSA4 and SSB2; SSA4 and ECMlO; SSA4 and MDJl; SSA4 and MDJ2; SSA4 and EROl; SSA4 and ERV2; SSA4 and EUGl; SSA4 and MPDl; SSA4 and MPD2; SSA4 and EPSl; SSA4 and PDIl; SSA4 and DERI; SSA4 and DER3; SSA4 and HRD3; SSA4 and UBC7; SSA4 and DOA4; SSEl and SSE2; SSEl and SSBl; SSEl and SSB2; SSEl and ECMlO; SSEl and MDJl; SSEl and MDJ2; SSEl and EROl; SSEl and ERV2; SSEl and EUGl; SSEl and MPDl; SSEl and MPD2; SSEl and EPSl; SSEl and PDIl; SSEl and DERI; SSEl and DER3; SSEl and HRD3; SSEl and UBC7; SSEl and DOA4; SSE2 and SSBl; SSE2 and SSB2; SSE2 and ECMlO; SSE2 and MDJl; SSE2 and MDJ2; SSE2 and EROl; SSE2 and ERV2; SSE2 and EUGl; SSE2 and MPDl; SSE2 and MPD2; SSE2 and EPSl; SSE2 and PDIl; SSE2 and DERI; SSE2 and DER3; SSE2 and HRD3; SSE2 and UBC7; SSE2 and DOA4; SSBl and SSB2; SSBl and ECMlO; SSBl and MDJl; SSBl and MDJ2; SSBl and EROl; SSBl and ERV2; SSBl and EUGl; SSBl and MPDl; SSBl and MPD2; SSBl and EPSl; SSBl and PDIl; SSBl and DERI; SSBl and DER3; SSBl and HRD3; SSBl and UBC7; SSBl and DOA4; SSB2 and ECMlO; SSB2 and MDJl; SSB2 and MDJ2; SSB2 and EROl; SSB2 and ERV2; SSB2 and EUGl; SSB2 and MPDl; SSB2 and MPD2; SSB2 and EPSl; SSB2 and PDIl; SSB2 and DERI; SSB2 and DER3; SSB2 and HRD3; SSB2 and UBC7; SSB2 and DOA4; ECMl 0 and MDJl ; ECMl 0 and MD J2; ECMl 0 and ERO 1 ; ECMl 0 and ERV2; ECMlO and EUGl; ECMlO and MPDl; ECMlO and MPD2; ECMlO and EPSl; ECMlO and PDIl; ECMlO and DERI; ECMlO and DER3; ECMlO and
HRD3; ECMlO and UBC7; ECMlO and DOA4; MDJl and MDJ2; MDJl and EROl; MDJl and ERV2; MDJl and EUGl; MDJl and MPDl; MDJl and MPD2; MDJl and EPSl; MDJl and PDIl; MDJl and DERI; MDJl and DER3; MDJl and HRD3; MDJl and UBC7; MDJl and DOA4; MDJ2 and EROl; MDJ2 and ERV2; MDJ2 and EUGl; MDJ2 and MPDl; MDJ2 and MPD2; MDJ2 and EPSl; MDJ2 and PDIl; MDJ2 and DERI; MDJ2 and DER3; MDJ2 and HRD3; MDJ2 and UBC7; MDJ2 and D0A4; EROl and ERV2; EROl and EUGl; EROl and MPDl; EROl and MPD2; EROl and EPSl; EROl and PDIl; EROl and DERI; EROl and DER3; EROl and HRD3; EROl and UBC7; EROl and D0A4; ERV2 and EUGl; ERV2 and MPDl; ERV2 and MPD2; ERV2 and EPSl; ERV2 and PDIl; ERV2 and DERI; ERV2 and DER3; ERV2 and HRD3; ERV2 and UBC7; ERV2 and D0A4; EUGl and MPDl; EUGl and MPD2; EUGl and EPSl; EUGl and PDIl; EUGl and DERI; EUGl and DER3; EUGl and HRD3; EUGl and UBC7; EUGl and D0A4; MPDl and MPD2; MPDl and EPSl; MPDl and PDIl; MPDl and DERI; MPDl and DER3; MPDl and HRD3; MPDl and UBC7; MPDl and D0A4; MPD2 and EPSl; MPD2 and PDIl; MPD2 and DERI; MPD2 and DER3; MPD2 and HRD3; MPD2 and UBC7; MPD2 and D0A4; EPSl and PDIl; EPSl and DERI; EPSl and DER3; EPSl and HRD3; EPSl and UBC7; EPSl and D0A4; PDIl and DERI; PDIl and DER3; PDIl and HRD3; PDIl and UBC7; PDIl and D0A4; DERI and DER3; DERI and HRD3; DERI and UBC7; DERI and D0A4; DER3 and HRD3; DER3 and UBC7; DER3 and DOA4; HRD3 and UBC7; HRD3 and D0A4; or UBC7 and D0A4.
PROTEIN PRODUCT OF CHOICE
In principle, any protein can be expressed as the protein product of choice.
As discussed above, the protein product of choice may or may not be a protein that is naturally produced by the host cell, in which case the protein may or may not be encoded by the host cell's endogenous gene for that protein or the protein may or may not be encoded (fully, or in part) by an exogenous polynucleotide sequence.
Thus, it is possible to produce enhanced levels of naturally produced proteins by transforming the host cell with a polynucleotide encoding a further, or replacement, copy of an endogenous gene, or otherwise genetically modifying the host cell to increase the expression of a naturally produced protein. In one embodiment, a recombinant or genetically modified endogenous gene has a sequence that is different to the endogenous genetic material of the host cell.
The protein product of choice may or may not be a heterologous protein, by which we mean that the protein is one that is not naturally produced by the host cell. In the case of a heterologous protein product of choice, the protein may or may not be encoded by an exogenous polynucleotide sequence.
In one embodiment, the protein product of choice 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 that encodes the protein product of choice.
Alternatively, the protein product of choice may or may not be intracellular.
It is known in the prior art that enhanced protein production can be achieved by co-expression of a protein product and a chaperone in different compartments of the cell. For example, WO 2005/061718 (Example 12) describes the co-over- expression of the cytoplasmic chaperone SSAl and a secreted recombinant transferrin, in order to increase the production of the secreted .recombinant transferrin.
In another preferred embodiment, the protein product of choice comprises the sequence of a eukaryotic protein, or a fragment or variant thereof. Suitable eukaryotes include fungi, plants and animals. In one preferred embodiment the protein product of choice is a fungal protein, such as a yeast protein. In another preferred embodiment the protein product of choice is an animal protein. Exemplary animals include vertebrates and invertebrates. Exemplary vertebrates include mammals, such as humans, and non-human mammals. Thus the protein product of choice may or may not comprise the sequence of a yeast protein.
The protein product of choice may or may not comprise 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-baseda 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 aprotinin, amyloid precursor protein and those described hi WO 03/066824, with or without albumin fusions), interferons (such as interferon α species and sub-species, interferon β species and sub-species, interferon γ species and sub-species), interleukins (such as ILlO, ILIl and IL2), leptin, CNTF and fragment thereof (such as CNTFAχi5\Axokine™)), ILl-receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20 peptide, T1249 peptide, HFV gρ41, HFV 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, Ot1 -antitrypsin, plasminogen activators, Factor VII, Factor VIII, Factor FX, Factor X and Factor XIII, nerve growth factor, LACI, platelet-derived endothelial cell growth factor (PD-ECGF), glucose oxidase, serum cholinesterase, inter-alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, 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 VaI3 He, Leu, Ala, Met; Asp, GIu; Asn, GIn; Ser, Thr, GIy5 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 firom 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 a!., (1994) Nucleic Acids Res., 22(22), 4673-80). The parameters used may be as follows:
• Fast pairwise alignment parameters: Kτtuple(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 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 protein product of choice 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. Preferably 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 PO 1012), 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 089020), 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, AU 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-stabiUty) 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 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 et al., 1994). The parameters used may 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.
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), 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. A fragment will 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, IIIA and IIIB). Sub- domain IA comprises amino acids 6-105, sub-domain IB comprises amino acids 120-177, sub-domain ILA comprises amino acids 200-291, sub-domain HB comprises amino acids 316-369, sub-domain IIIA comprises amino acids 392-491 and sub-domain IIIB 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 protein product of choice 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, VCH5 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. Purif, 8, 119; Mason et al, 1991, Protein Expr. Purif., 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. Set USA, 92, 2820; AIi et al, 1999, J Biol. Chem., 21 A, 24066; Mason et al, 2002, Biochemistry, 41, 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 immunoglobulins or a variant or fragment of any of these may 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-terrninal 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 U S A, 92, 2820, AIi, et al., 1999, J Biol Chem, 274, 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 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 be presented alone or as fusion partners for albumin, the Fc region of immunoglobulin, transferrin, lactoferrin or any other protein or fragment or variant of any of the above, as a desired polypeptide.
The protein product of choice 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.
As discussed above, the protein product of choice may or may not comprise a leader sequence effective to cause secretion in the host cell (such as in a yeast host 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 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 eukaryotic species such as the yeasts Saccharomyces cerevisiae > Zygosaccharomyces species, Kluyveromyces lactis and Pichia pastoris, 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 the examples of this application and 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 protein products of choice.
Where a helper protein is a chaperone involved in the formation of disulphide bonds, then in one embodiment the protein product of choice comprises disulphide bonds in its mature form. The disulphide bonds may be intramolecular and/or mtermolecular.
The protein product of choice may or may not be a commercially useful protein. Some 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 as a protein, but that that effect is not the main or sole reason for expressing the protein therein.
SUITABLE HOST CELLS FOR THE PRACTICE OF THE PRESENT INVENTION
The host cell may be any type of cell. The host cell may or may not be an animal (such as mammalian, avian, insect, etc.), plant, fungal or bacterial cell. Bacterial and fungal, such as yeast, host cells may or may not be preferred.
Thus, the host cell may or may not be an animal (such as mammalian, avian, insect, etc.) cell. Suitable methods for transformation of animal cells are well known in the art and include, for example the use of retrovirus vectors (such as lentivirus vectors). Wolkowicz et al, 2004, Methods MoI. Biol, 246, 391-411 . describes the use of lentivirus vectors for delivery of recombinant nucleic acid sequences to mammalian cells for use in cell culture techniques. Fassler, 2004, EMBO Rep., 5(1), 28-9 reviews lentiviral transgene vectors and their use in the production of transgenic systems.
In one embodiment the host cell is a yeast cell, such as a member of the Saccharomyces, Kluyveromyces, or Pichia genus, such as Saccharomyces cerevisiae, Kluyveromyces lactis, Pichia pastoris and Pichia membranaefaciens, or Zygosaccharomyces rouxii, Zygosaccharomyces bailii, Zygosaccharomyces fermentati, Hansenula polymorpha (also known as Pichia angusta) or Kluyveromyces drosophilarum are preferred.
It may 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 T/GB2006/002289 reported to be modified, in nature, by O-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 hi 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. Alternatively, the yeast could be cultured in the presence of a compound that inhibits the activity of one of the PMT genes (Duffy et al, "Inhibition of protein mannosyltransf erase 1 (PMTl) activity in the pathogenic yeast Candida albicans", International Conference on Molecular Mechanisms of Fungal Cell Wall Biogenesis, 26-31 August 2001, Monte Verita, Switzerland, Poster Abstract P38; the poster abstract may be viewed at http://www.micro.biol.ethz.ch/cellwall/).
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. hi Pichia pastoris or Kluyverojnyces lactis. The sequence of PMTl (or any other PMT gene) isolated from S. cerevisiae may 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 also have a deletion of the HSP 150 and/or YAP 3 genes as taught respectively in WO 95/33833 and WO 95/23857. Where one or more of Hie helper protein(s) and/or protein product of choice axe encoded by a plasmid-borne polynucleotide sequence, the host cell type may be selected for compatibility with the plasmid type being used.
The skilled person will appreciate that any suitable plasmid may be used, such as a centromeric plasmid. The examples provide suitable plasmids (centromeric YCplac33-based vectors) for use to transform yeast host cells of the present invention. Alternatively, any other suitable plasmid may be used, such as a yeast- compatible 2μm-based plasmid.
Plasmids obtained from one yeast type can be maintained in other yeast types (Irie et al, 1991, Gene, 108(1), 139-144; Irie et al, 1991, MoI. Gen. Genet, 225(2), 257-265). For example, pSRl from Zygosaccharomyces rouxii can be maintained in Saccharomyces cerevisiae. In one embodiment the plasmid may or may not be a 2μm-family plasmid and the host cell will be compatible with the 2μm-family plasmid used (see below 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 pELDl 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. Thus, the plasmid may be based on the 2μm plasmid and the yeast cell may be Saccharomyces cerevisiae. A 2μm~famiry plasmid can be said to be "based on" a naturally occurring plasmid if it comprises one, two or preferably three of the genes FiP, REPl and REP2 having sequences derived from that naturally occurring plasmid.
A plasmid as defined above, may be introduced into a host through standard techniques. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl Acad. ScI USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3rd 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 Laboratory 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 251 744, 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 fungal (including yeast) cell, plant cells, bacterial cells and animal (including vertebrate) cells. Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol 194, 182.
Generally, the plasmid will transform not all of the hosts and it will therefore be necessary to select for transformed host cells. Thus, a plasmid may 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. Typical yeast selectable marker include LEU2, TRPl, HIS3, HIS4, URA3, URA5, SFAl, ADE2, METIS, LYS5, LYS2, ILV2, FBAl, PSEl, PDU and PGKl. Those skilled in the art will appreciate that any gene whose chromosomal deletion or inactivation results in an unviable 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, 119). Suitable essential genes can be found within the Stanford Genome Database (SGD), (http:://db.yeastgenome.org). Any essential gene product (e.g. PDIl, PSEl, PGKl or FBAl) 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 additions or modifications to the growth medium. Therefore, preferred "essential marker genes" in the context of the present application are those that, when deleted or inactivated hi a host cell, result in a deficiency which cannot be complemented by additions or modifications to the growth medium. Additionally, a plasmid may 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, neomycin or zeocin resistance for eukaryotic cell culture, and tetracycline, kanamycin, ampicillin (i.e. β-lactamase) or zeocin resistance genes for culturing in E. coli and other bacteria. Zeocin resistance vectors are available from Invitrogen. 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 a plasmid, optionally to allow the expression of a recombinant polypeptide (i.e. a polypeptide which is encoded by a polynucleotide sequence on the plasmid and is heterologous to the host cell, in the sense that that polypeptide 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 MoL Biol. 98, 503 or Berent et al (1985) Biotech. 35 208 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 assaying 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, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium. Alternatively, transformed cells may 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.
Transformed host cells may 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 the helper protein(s) and the protein product of choice.
The culture medium may be non-selective or place a selective pressure on the maintenance of a plasmid.
The thus produced protein product of choice may be present intracellularly or, if secreted, in the culture medium and/or periplasmic space of the host cell.
Accordingly, the present invention. also provides a method for producing a protein product of choice, the method comprising: (a) providing a host cell of the invention comprising a polynucleotide encoding protein product of choice as defined above; and
(b) growing the host cell (for example, culturing the host cell in a culture medium);
thereby to produce a cell culture or recombinant organism comprising an increased level of the protein product of choice compared to the level of production of the protein product of choice achieved by growing (for example, culturing), under the same conditions, the same host cell that has not been genetically modified to cause over-expression of one or more helper proteins.
The step of growing the host cell may or may not involve allowing a host cell derived from a multicellular organism to be regrown into a multicellular recombinant organism (such as a plant or animal) and, optionally, producing one or more generations of progeny therefrom.
The method may or may not further comprise the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.
The step of "purifying the -thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium" optionally comprises cell immobilisation, cell separation and/or cell breakage, but always comprises at least one other purification step different from the step or steps of cell immobilisation, separation and/or breakage.
Cell immobilisation 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, hydroxyapatite chromatography, lectin chromatography, concentration, dilution, pH adjustment, diafiltration, ultrafiltration, high performance liquid chromatography ("HPLC"), reverse phase HPLC5 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 purifying 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 lO^g.L4, lO"3 g.L4 5 0.01 gl/1, 0.02 gX4, 0.03 g.L4, 0.04 g.L4, 0.05 g.L4, 0.06 gX4,0.07 g.L4, 0.08 g.L4, 0.09 g.L4, 0.1 g.L4, 0.2 g.L4, 0.3 g.L4, 0.4 gX4, 0.5 gX4, 0.6 g.L4, 0.7 glΛ 0.8. gX4, 0.9 g.L"1, 1 g.L4, 2 gX4, 3 g.L4, 4 gX4, 5 gX"1, 6 g.L4, 7 g.L4, 8
Figure imgf000159_0001
9 g.L4, 10 g.L4, 15 g.L4, 20 g.L4, 25 gX4, 30 gX4, 4.0 g.L4,50 g.L4, 60 g.L4, 70 gX4, 70 g.L4, 90 g.L4, 100 g.L4, 150 g.L4, 200 g.L4, 250 g.L4, 300 g.L4, 350 g.L4, 400 g.L4, 500 g.L4, 600 g.L4, 700 g.L4, 800 g.L4, 900 g.L4, 1000 g.L4, or more. B2006/002289
A commercially or industrially acceptable level of purity may be obtained by a relatively crude purification method by which the protein product of choice is put into a form suitable for its intended purpose. A protein preparation that has been purified to a commercially or industrially acceptable level of purity may, in addition to the protein product of choice, also comprise, for example, cell culture components such as host cells or debris derived therefrom. Alternatively, high molecular weight components (such as host cells or debris derived therefrom) may or may not be removed (such as by filtration or centrifugation) to obtain a composition comprising the protein product of choice and, optionally, a functionally acceptable level of low molecular weight contaminants derived from the cell culture process.
The protein may or may not be 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 protein may be used for any of its known utilities, which, in the case of albumin, include i.v. administration to patients to treat severe burns, shock and blood loss, supplementing culture media, and as an excipient in formulations of other proteins.
A method of the present invention may or may not further comprise the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
Although it is possible for a therapeutically useful protein obtained by a process of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, 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 not deleterious to the recipients 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.
Alternatively, a method of the present invention may or may not further comprise the step of lyophilising the thus purified protein product of choice.
DETAILED DESCRIPTION OF HELPER PROTEINS
JEMl is one S. cerevisiae helper protein of interest for the present invention. It is also known as KAR8, and its gene is a non-essential gene located on chromosome X. It is a DnaJ-like chaperone and is thought to be required for nuclear membrane fusion during mating. It localises to the ER membrane and exhibits genetic interactions with Kar2p (described further below). A published protein sequence for the protein Jemlp is as follows:
MILISGYCLLVYSVILPVLISASKLCDLAELQRLNKNLKVDTESLPKYQWIAGQLEQNCM TADPASENMSDVIQLANQIYYKIGLIQLSNDQHLRAINTFEKIVFNETYKGSFGKLAEKR LQELYVDFGMWDKVHQKDDQYAKYLSLNETIRNKISSKDVSVEEDISELLRITPYDVNVL STHIDVLFHKLAEEIDVSLAAAIILDYETILDKHLASLSIDTRLSIHYVISVLQTFVLNS DASFNIRKCLSIDMDYDKCKKLSLTISKLNKVNPSKRQILDPATYAFENKKFRSWDRIIE FYLKDKKPFITPMKILNKDTNFKNNYFFLEEIIKQLIEDVQLSRPLAKNLFEDPPITDGF VKPKSYYHTDYLVYIDSILCQASSMSPDVKRAKLAAPFCKKSLRHSLTLETWKHYQDAKS EQKPLPETVLSDVWNSNPHLLMYMVNSILNKSRSKPHSQFKKQLYDQINKFFQDNGLSES TNPYVMKNFRLLQKQLQTYKEHKHRNFNQQYFQQQQQQQQHQRHQAPPAAPNYDPKKDYY • KILGVSPSASSKEIRKAYLNLTKKYHPDKIKANHNDKQESIHETMSQINEAYETLSDDDK RKEYDLSRSNPRRNTFPQGPRQNNMFKNPGSGFPFGNGFKMNFGL*
The ORF ofthe JEMl gene is 1.938 kbp in size. A published nucleotide coding sequence ofJEMl is as follows, although it will be appreciated thatthe sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
ATGATACTGATCTCGGGATACTGTCTTTTAGTGTATAGCGTTATTTTGCCAGTACTGATA TCGGCTTCTAAGTTATGTGATTTGGCTGAGTTACAACGATTGAACAAGAATTTAAAAGTA GACACTGAATCCTTGCCAAAATACCAATGGATCGCTGGGCAGTTGGAACAAAΆCTGCATG ACTGCGGATCCAGCAAGTGAAAATATGTCAGACGTAATTCAACTAGCCAATCAAΆTATAC TACAAAATTGGGCTGATCCAATTATCCAACGATCAACΆTCTAAGAGCTATTAACACATTT GAAAAAATCGTTTTTAATGAAACTTACAAAGGTTCTTTTGGGAAGCTGGCGGAAΆAGAGG CTACAAGAGCTGTATGTCGATTTTGGGATGTGGGACAAGGTGCATCAGAAGGATGATCAG TATGCGAAATATCTGTCCTTGAATGAAACCATCAGAAΆCAAAATATCATCCAAAGACGTT TCTGTGGAGGAAGATATTTCTGAGCTGCTACGCATAACGCCGTACGATGTTAACGTCCTC TCCACGCACATCGATGTTCTTTTTCACAAACTAGCTGAAGAΆATTGACGTTTCGTTAGCT GCTGCTATCATTTTGGATTACGAAACAATCCTCGACAAGCATTTGGCTAGCTTAAGCATA GATACAAGACTTTCGATTCATTATGTCATATCTGTTTTACAGACCTTTGTACTTAACTCA GATGCGTCGTTCAATATAAGAAAATGCCTTTCCATTGATATGGACTATGATAAATGTAAΆ AAACTAAGCCTGACTATTTCCAAATTGAACAAGGTGAATCCATCAAAΆAGACAGATCCTG GΆTCCAGCAACATATGCATTTGAGAACAAAAAGTTTAGAAGTTGGGATAGAATTATTGAA TTTTATTTGAΆGGATAAGAAGCCATTTATTACACCAATGAAAATTCTTAACAAAGATΆCA AACTTTAAAAACAACTACTTCTTTTTAGAGGΆAATTATCAΆΆCAATTGATAGAΆGACGTT CAΆCTGTCGAGACCTTTGGCAAAAΆATTTATTCGAAGΆTCCCCCAATAACCGATGGTTTT GTCAAACCAAΆATCATACTATCATACCGATTATCTAGTATACATTGATTCCATTCTTTGT CAGGCTTCTAGCATGAGTCCGGACGTCAAGAGAGCTAAACTGGCTGCGCCGTTCTGTAAA AAGAGTTTGAGGCATTCACTAΆCACTAGAΆΆCATGGAAΆCACTATCAGGATGCTAΆGTCC GAGCAAAAACCTTTACCTGAGACGGTATTGAGTGATGTATGGAATTCCAATCCTCATTTG CTGATGTATATGGTAAACTCAATACTTAATAAAAGTAGGTCTAAACCTCATTCACAGTTC AAAAΆGCAATTATATGACCAGATAAACAAATTTTTCCAAGATAACGGCCTCTCAGAGTCG ACCAATCCATACGTGATGAAGAACTTCCGATTATTACAGAAACAΆTTACAAΆCCTΆTAAA GAGCATAAACATCGGAATTTCAACCAGCAATATTTCCAACAACAACΆΆCAGCAGCAACAA CACCAACGACATCAAGCACCCCCAGCAGCGCCTAACTACGACCCAAAAAAGGACTATTAT AAAATTCTTGGAGTATCGCCTAGTGCTAGTTCGAΆΆGAΆATAAGGAAAGCATATTTAAAT TTAACCAAAAAΆTACCACCCAGACAAAATAAAGGCCAACCATAACGΆCAAACAAGAATCA ATTCACGAAACTATGTCACAAΆTCAATGAAGCGTACGAAΆCATTAΆGTGATGACGATAAA AGGAAGGAATACGATCTTTCCAGATCAAACCCCCGCCGCAACACTTTTCCTCAGGGGCCT AGGCAAAATAACATGTTCAAAAATCCAGGAAGTGGCTTCCCATTCGGAAATGGCTTTAAA ATGAATTTTGGGCTTTGA
Further information concerning JEMl can be seen at the following URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000003609.
It will be appreciated that, by "JEMl", we include fragments or variants thereof having equivalent JEMl-like activity. Such variants may or may not include bacterial DnaJ proteins and/or may or may not include eukaryotic DnaJ type 2006/002289
proteins, such as other members of the Hsp40 family. In one embodiment, a variant of JEMl may not be SCJl.
LHSl is another S. cerevisiae helper protein of interest for the present invention. It is also known as CERl or SSIl, is encoded by a non-essential gene which is located on chromosome XI. It is thought to be a molecular chaperone of the endoplasmic reticulum lumen, involved in polypeptide translocation and folding. It is a member of the HSP70 family, localizes to the lumen of the ER, and is thought to be regulated by the unfolded protein response pathway.
A published protein sequence for the protein Lhslp is as follows:
MRNVLRLLFLTAFVAIGSLAAVLGVDYGQQNIKAIWSPQAPLELVLTPEAKRKEISGLS IKRLPGYGKDDPNGIERIYGSAVGSLATRFPQNTLLHLKPLLGKSLEDETTVTLYSKQHP GLEMVSTNRSTIAFLVDNVEYPLEELVAMNVQEIANRANSLLKDRDARTEDFVNKMSFTI PDFFDQHQRKALLDASSITTGIEETYLVSEGMSVAVNFVLKQRQFPPGEQQHYIVYDMGS GSIKASMFSILQPEDTTQPVTIEFEGYGYNPHLGGAKFTMDIGSLIENKFLETHPAIRTD ELHANPKALAKINQAAEKAKLILSANSEASINIESLINDIDFRTSITRQEFEEFIADSLL DIVKPINDAVTKQFGGYGTNLPEINGVILAGGSSRIPIVQDQLIKLVSEEKVLRNVNΆDE SAVNGVVMRGIKLSNSFKTKPLNWDRSVNTYSFKLSNESELYDVFTRGSAYPNKTSILT NTTDSIPNNFTIDLFENGKLFETITVNSGAIKNSYSSDKCSSGVAYNITFDLSSDRLFSI QEVNCICQSENDIGNSKQIKNKGSRLAFTSEDVEIKRLSPSERS RLHEHIKLLDKQDKER FQFQENLNVLEΞNLYDARNLLMDDEVMQNGPKΞQVEELSEMVKVYLDWLEDASFDTDPED IVSRIREIGILKKKIELYMDSAKEPLNSQQFKGMLEEGHKLLQAIETHKNTVEEFLSQFE TEFADTIDNVREEFKKIKQPAYVSKALSTWEETLTSFKNSISEIEKFLAKNLFGEDLREH LFEIKLQFDMYRTKLEEKLRLIKSGDESRLNEIKKLHLRNFRLQKRKEEKLKRKLEQEKS RNNNETESTVINSADDKTTIVNDKTTESNPSSEEDILHDEL*
The ORF of the LHSl gene is 2.646 kbp .in size. A published nucleotide coding sequence of LHSl is as follows, 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:
ATGCGAAACGTTTTAAGGCTTTTATTTTTAACAGCTTTTGTTGCTATAGGGTCTTTAGCA GCCGTTTTAGGTGTTGATTACGGTCAGCAAAATATCAAGGCCATTGTGGTTTCTCCGCAA
GCCCCATTAGAACTTGTGCTCACACCAGAGGCAAAACGGAAGGAGATATCTGGTCTTTCG B2006/002289
ATAAΆAAGATTACCAGGTTATGGAΆAGGATGATCCGAATGGGATTGAAΆGAATCTACGGT TCCGCTGTTGGCAGTTTAGCAACAAGGTTTCCCCAAAACACATTGTTGCATTTGAΆACCG CTACTTGGGAAATCACTAGAAGATGAAACCACTGTAACTTTGTATTCAAAACAACACCCC GGTTTAGAAATGGTATCAACAAATAGAAGTACCATAGCCTTTTTAGTTGATAATGTGGAA TATCCATTGGAAGAGTTAGTGGCAATGAATGTCCAAGAGATTGCCAATAGAGCCAATTCA CTGTTGAAGGATAGAGATGCAAGAACTGAGGACTTTGTAAACAAGATGAGTTTTACAATT CCTGACTTTTTTGACCAACATCAΆAGGAAAGCACTTTTAGATGCCΆGTTCAATAΆCCACA GGAATCGAAGAGACATATCTGGTTAGTGAAGGGATGTCTGTTGCAGTTAACTTTGTATTA AAGCAGCGCCAATTTCCACCAGGTGAACAGCAGCATTATATCGTATATGACATGGGGAGC GGTTCTATTAAGGCCTCAATGTTCTCTATATTGCAGCCGGAGGACACTACTCAGCCCGTT ACAATAGAATTTGAAGGATATGGGTATAATCCACATCTAGGTGGTGCAAAGTTTACAATG GATATTGGCAGTTTGATAGAGAATAAGTTTTTGGAAACACACCCAGCCATAAGAACTGAT GAATTGCACGCTAΆTCCCAAGGCCTTAGCAAAAΆTCAACCAAGCAGCAGAGAAGGCAAAG TTAATTTTAAGCGCCAATTCTGAGGCAAGTATTAΆCATAGAATCACTGATCΆACGATATT GATTTCCGTACTTCTATAACTAGACAGGAATTCGAAGAATTTATTGCAGACTCGTTATTG GACATTGTCAAACCCATAAATGACGCTGTTACAΆAACAATTCGGTGGCTATGGAΆCAAAT TTACCTGAGATAAATGGGGTCATTTTGGCGGGΆGGCTCTTCCCGAATTCCCATTGTGCAG GATCAATTAATCAAΆCTCGTATCCGAAGAAΆΆAGTGTTGAGAAATGTCAATGCTGATGAA TCAGCTGTGAATGGTGTTGTTATGAGAGGGATCAAGTTATCTAATTCGTTTAAGACCAAG CCGTTAAATGTTGTTGACCGTTCTGTAAATACTTATTCATTCAAATTATCAAACGAATCT GAACTGTATGATGTGTTCACGCGCGGAAGTGCTTATCCAAACAAAACATCTATTTTGACA AACΆCGACTGATTCGATTCCTAATAATTTTACCATTGACTTATTTGAGAΆTGGTAAΆTTG TTCGAAACTATCACAGTTAATTCAGGAGCTATAAAGAATTCATATTCCTCTGATAAGTGC TCGTCAGGAGTTGCGTATAACATTACTTTCGACTTGTCCAGTGATAGATTATTCTCTATT CAAGAGGTTAACTGCATTTGTCAGAGCGAAAATGACATAGGTAACTCCAΆGCAΆΆTTAAG AACAAAGGCAGCCGTTTGGCTTTTACTTCTGAGGATGTTGAGATCAAAAGGCTTTCTCCT TCAGAACGTTCGCGTTTGCATGAGCATATCAAGTTGCTCGATAΆACAGGATAΆGGAAΆGA TTTCAATTCCAAGAAAATTTAAACGTTCTTGAAΆGTAACTTGTATGATGCTAGAΆACCTG CTAATGGATGATGAAGTTATGCAAAATGGACCAAAΆTCCCAAGTAGAAGAGTTATCGGAG ATGGTTAAAGTATATTTGGATTGGCTCGAAGATGCATCCTTTGATACTGACCCTGAGGAT ATAGTTAGCAGAATTAGAGAAATTGGAATATTAAAAAAGAAAATAGAACTTTACATGGAT TCTGCAΆAGGAACCTTTGAACTCTCAΆCΆATTTAAΆGGAATGCTTGAAGAAGGCCATAAG TTACTTCAGGCTATAGΆAACCCATAAGAATACCGTTGAAGAATTTTTGAGTCAΆTTTGAA ACCGAGTTTGCGGATACCATAGATAATGTTAGAGAAGAATTTAAAAAGATTAAGCAACCA GCGTATGTGTCGAAGGCGTTATCTACATGGGAGGAAACCTTAACCTCTTTTAAAAATTCC ATTΆGCGAAΆTAGAGAΆGTTCCTGGCAAAΆAACCTATTTGGCGAAGACCTTCGTGAACAT TTATTTGAAATCAAΆTTACAATTTGATATGTATCGTACGAAΆCTAGAGGAΆAAACTGCGT TTAATAAAAΆGCGGTGATGAAAGTCGCTTAAATGAAATAAAGAAGTTACATTTAAGAAAC TTCCGCCTACAAAAGAGAAAGGAGGAAAAGTTGAAΆAGAΆAGCTTGAΆCAGGAAAAAAGC AGAAACAACAATGAAACAGAATCGACAGTAATCAACTCGGCTGACGATAAAACTACTATT B2006/002289
GTCAATGACAAGΆCCACCGAGTCGAATCCAAGTTCTGAGGAAGACATTTTGCATGATGAA
TTATAG
Further information on LHSl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000001556.
It will be appreciated that, by "LHSl", we include fragments or variants thereof having equivalent LHSl -like activity. Such variants may or may not include bacterial DnaK proteins and/or eukaryotic DnaK type proteins, such as other members ofthe Hsp70 family.
SCJl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of bacterial chaperone DnaJ, located in the ER lumen where it cooperates with Kar2p (described below) to mediate maturation of proteins.
A published protein sequence for the protein Scj Ip is as follows:
MIPKLYIHLILSLLLLPLILAQDYYAILEIDKDATEKEIKSAYRQLSKKYHPDKNAGSEE AHQKFIEVGEAYDVLSDPEKKKIYDQFGADAVKNGGGGGGPGGPGAGGFHDPFDIFERMF
QGGHGGPGGGFGQRQRQRGPMIKVQEKLSLKQFYSGSSIEFTLNLNDECDACHGSGSADG
KLAQCPDCQGRGVIIQVLRMGIMTQQIQQMCGRCGGTGQIIKNECKTCHGKKVTKKNKFF
HVDVPPGAPRNYMDTRVGEAEKGPDFDAGDLVIEFKEKDTENMGYRRRGDNLYRTEVLSA
AEALYGGWQRTIEFLDENKPVKLSRPAHVWSNGEVEWKGFGMPKGSKGYGDLYIDYW VMPKTFKSGQNMLKDEL*
SCJl is encoded by a non-essential gene comprising an ORF of 1.134 kbp. The gene is located on chromosome XIII. A published nucleotide coding sequence of SCJl is as follows, 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:
ATGATTCCAAAATTATATATACATTTGATACTATCTTTATTGTTGTTGCCGCTAATTTTG
GCGCAGGATTATTATGCAATACTAGAGATAGACAAAGATGCCACTGAGAAGGAAATCAAA TCAGCGTACAGACAATTGTCTAAGAAGTACCATCCGGATAAAAATGCTGGGAGCGAAGAA GCCCATCAAAAATTCATTGAAGTCGGCGAGGCATACGATGTATTGAGCGATCCTGAAAAG AAAAAGATTTATGACCAGTTTGGTGCAGATGCTGTAAAGAATGGCGGTGGCGGTGGCGGT CCAGGAGGCCCTGGCGCAGGTGGATTCCACGATCCGTTTGACATATTCGAACGGATGTTT ' CAAGGAGGTCATGGAGGTCCTGGCGGCGGATTTGGCCAGAGACAGAGGCAGCGTGGTCCA ATGATCAAGGTCCAGGAAAAACTATCTTTAAAGCAGTTTTATTCCGGGTCCTCGATAGAA TTTACTTTAAACCTAAACGATGAATGTGATGCATGCCATGGTAGTGGCTCTGCAGATGGT AAGCTGGCCCAΆTGTCCCGATTGTCAΆGGTCGTGGGGTTATAATACAAGTGCTGCGCATG GGTATTATGACGCAGCAGATTCAACAGATGTGTGGTAGGTGTGGTGGTACGGGACAAATT ATCAAAAATGAATGCAAAACATGTCACGGCAAAΆAAGTTACCAAAAAGAΆCAAGTTCTTC CACGTTGACGTTCCACCAGGCGCACCAAGAAACTACATGGACACAAGAGTCGGCGAGGCT GAAAAAGGGCCTGACTTTGACGCCGGTGACTTGGTCATAGAATTCAAGGAAΆAGGATACT GAGAACATGGGTTACAGAAGAAGAGGCGACAATCTGTACAGAACAGAAGTTCTTTCTGCT GCGGAAGCGCTATACGGCGGATGGCAAAGAACGATAGAATTCCTTGATGAGAACAAGCCC GTTAAGTTATCTAGACCCGCTCATGTAGTTGTCTCCAATGGCGAAGTTGAΆGTCGTGAAG GGATTCGGCATGCCCAAGGGTAGCAAGGGTTACGGTGATTTGTACATAGACTACGTCGTT GTCATGCCAAAGACTTTCAAATCTGGGCAAAATATGCTCAAAGATGAGTTGTAG
Further information on SCJl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000004827.
It will be appreciated that, by "SCJl", we include fragments or variants thereof having equivalent SCJl -like activity.
KAR2 is another S. cerevisiae helper protein of interest for the present invention. KAR2 is also known as BIP or GRP78. Kar2p, is an ATPase involved in protein import into the ER. Kar2p also acts as a chaperone to mediate protein folding in the ER and may play a role in ER export of soluble proteins. It is also thought to regulate the unfolded protein response via interaction with Irelp. A published protein sequence for the protein Kar2p is as follows: . •
MFFNRLSAGKLLVPLSWLYAL FWILPLQNSFHSSNVL VRGADDVENYGTVIGIDLGTT
YSCVAVMKNGKTEILANEQGNRITPSYVAFTDDERLIGDAAKNQVAANPQNTIFDIKRLI GLKYNDRSVQKDIKHLPFNWNKDGKPAVEVSVKGEKKVFTPEEISGMILGKMKQIAEDY LGTKVTHAWTVPAYFNDAQRQATKDAGTIAGLNVLRIVNEPTAAAIAYGLDKSDKEHQI IVYDLGGGTFDVSLLSIENGVFEVQATSGDTHLGGEDFDYKIVRQLIKAFKKKHGIDVSD NNKΆLAKLKREAEKAKRALSSQMSTRIEIDSFVDGIDLSETLTRAKFEELNLDLFKKTLK PVEKVLQDSGLEKKDVDDIVLVGGSTRIPKVQQLLESYFDGKKASKGINPDEAVAYGAAV QAGVLSGEEGVEDIVLLDVNALTLGIETTGGVMTPLIKRNTAIPTKKSQIFSTAVDNQPT
VMIKVYEGERAMSKDNNLLGKFELTGIPPAPRGVPQIEVTFALDANGILKVSATDKGTGK SESITITNDKGRLTQEEIDRMVEEAEKFASEDASIKAKVESRNKLENYAHSLKNQVNGDL GEKLEEEDKETLLDAANDVLEWLDDNFETAIAEDFDEKFESLSKVAYPITSKLYGGADGS GAADYDDEDEDDDGDYFEHDEL*
KAR2 is encoded by an essential gene comprising an ORP that is 2.049 kbp in size and located on chromosome X. A published nucleotide coding sequence of KAR2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidentical proteinproduct:
ATGTTTTTCAACAGACTAAGCGCTGGCAAGCTGCTGGTACCACTCTCCGTGGTCCTGTAC GCCCTTTTCGTGGTAATATTACCTTTACAGAATTCTTTCCACTCCTCCAATGTTTTAGTT AGAGGTGCCGATGATGTAGAAAACTACGGAACTGTTATCGGTATTGACTTAGGTACTACT TATTCCTGTGTTGCTGTGATGAAAAATGGTAAGACTGAAATTCTTGCTAATGAGCAAGGT AACAGAATCACCCCATCTTACGTGGCATTCACCGATGΆTGAAAGATTGATTGGTGATGCT GCAAAGAACCAAGTTGCTGCCAATCCTCAAAACACCATCTTCGACATTAAGAGATTGATC GGTTTGAAATATAACGACAGATCTGTTCAGAAGGATATCAAGCACTTGCCATTTAATGTG GTTAΆTAAAGATGGGAAGCCCGCTGTAGAΆGTAAGTGTCAAΆGGAGAAΆAGAΆGGTTTTT ACTCCAGAΆGAAATTTCTGGTATGATCTTGGGTΆAGATGAAACAAATTGCCGAAGATTAT TTAGGCACTAAGGTTACCCATGCTGTCGTTACTGTTCCTGCTTATTTCAATGΆCGCGCAA AGACAΆGCCACCAAGGATGCTGGTACCATCGCTGGTTTGAΆCGTTTTGAGAΆTTGTTAΆT GAACCAACCGCAGCCGCCATTGCCTACGGTTTGGATAAATCTGΆTAΆGGAACATCAΆATT ATTGTTTATGATTTGGGTGGTGGTACTTTCGATGTCTCTCTATTGTCTATTGAAAACGGT GTTTTCGAΆGTCCAAGCCACTTCTGGTGATACTCATTTΆGGTGGTGAAGATTTTGACTAT AAGATCGTTCGTCAΆTTGATAAΆAGCTTTCAAGAAGAAGCATGGTATTGATGTGTCTGAC AACAACAAGGCCCTAGCTAAATTGAAGAGAGAAGCTGAAAAGGCTAAACGTGCCTTGTCC AGCCAAATGTCCACCCGTATTGAAATTGACTCCTTCGTTGATGGTATCGACTTAAGTGAA ACCTTGACCAGAGCTAAGTTTGAGGAATTAAACCTAGATCTATTCAAGAAGACCTTGAAG CCTGTCGAGAAGGTTTTGCAΆGATTCTGGTTTGGAΆAAGAAGGATGTTGATGATATCGTT TTGGTTGGTGGTTCTACTAGAATTCCAΆAGGTCCAACAATTGTTAGAΆTCATACTTTGAT GGTAAGAAGGCCTCCAΆGGGTATTAACCCAGATGAAGCTGTTGCATACGGTGCAGCCGTT CAAGCTGGTGTCTTATCCGGTGAAGAAGGTGTCGAAGATATTGTTTTATTGGATGTCAAC GCTTTGACTCTTGGTATTGAAACCACTGGTGGTGTCATGACTCCATTAATTAAGAGAAAT ACTGCTATTCCTACAAAGAΆATCCCAAATTTTCTCTACTGCCGTTGACAΆCCAACCAACC GTTATGATCAAGGTATACGAGGGTGAAAGAGCCATGTCTAAGGACAACAATCTATTAGGT AAGTTTGAATTAACCGGCATTCCACCAGCACCAAGAGGTGTACCTCAAATTGAAGTCACA T/GB2006/002289
TTTGCACTTGACGCTAATGGTATTCTGAAGGTGTCTGCCACAGATAAGGGAACTGGTAAA TCCGAATCTATCACCATCACTAACGATAAAGGTAGATTAACCCAAGAAGAGATTGATAGA ATGGTTGAAGAGGCTGAAAAATTCGCTTCTGAAGACGCTTCTATCAAGGCCAAGGTTGAA TCTAGAAACAAATTΆGAAAACTACGCTCACTCTTTGAAAΆACCAΆGTTAATGGTGACCTA GGTGAAΆAATTGGAAGAAGAΆGACAΆGGAAACCTTATTAGATGCTGCTAACGATGTTTTA GAATGGTTAGATGATAACTTTGΆAACCGCCATTGCTGAAGACTTTGΆTGAAΆAGTTCGAA TCTTTGTCCAAGGTCGCTTATCCAATTACTTCTAAGTTGTACGGAGGTGCTGATGGTTCT GGTGCCGCTGATTATGACGACGAAGATGAAGATGACGATGGTGATTATTTCGAACACGAC GAATTGTAG
Further information on KAR2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000003571.
It will be appreciated that, by "KAR2", we include fragments or variants thereof having equivalent KAR2-like activity.
SILl is another S. cerevisiae helper protein of interest for the present invention and is also known as SLSl. In particular, this helper protein was generally referred to as SLSl in UK patent application no. 0512707.1, from which this application claims priority; it will be understood by the person skilled in the art that reference in UK patent application no. 0512707.1 to SLSl and reference in this application to SILl should be taken to be reference to the same helper protein. SILIp is an ER-localized protein required for protein translocation into the ER, which interacts with the ATPase domain of the Kar2p chaperone suggesting some role in modulating its activity. It is also thought to be a homolog of Yarrowia lipofytica SILl; and a GrpE-like protein in the ER. A published protein sequence for the protein SILIp is as follows:
MVRILPIILSALSSKLVASTILHSSIHSVPSGGEIISAEDLKELEISGNSICVDNRCYPK IFEPRHDWQPILPGQELPGGLDIRINMDTGLKEAKLNDEKNVGDNGSHELIVSSEDMKAS
PGDYEFSSDFKEMRNIIDSNPTLSSQDIARLEDSFDRIMEFAHDYKHGYKIITHEFALLA
NLSLNENLPLTLRELSTRVITSCLRNNPPWEFINESFPNFKSKIMAALSNLNDSNHRSS
NILIKRYLSILNELPVTSEDLPIYSTWLQNVYERNNKDKQLQIKVLELISKILKADMYE
NDDTNLILFKRNAENWSSNLQEWANEFQEMVQNKSIDELHTRTFFDTLYNLKKIFKSDIT INKGFLNWLAQQCKARQSNLDNGLQERDTEQDSFDKKLIDSRHLIFGNPMAHRIKNFRDE
L* SILl is encoded by a non-essential gene comprising an ORP that is 1.226 kbp in size and is located on chromosome XV. A published nucleotide coding sequence of 5ZLi is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGGTCCGGATTCTTCCCATAATTTTGAGCGCCCTATCTTCGAAATTAGTGGCGAGTACA ATATTGCATTCATCCATACACTCAGTGCCATCTGGAGGCGAAATCATATCTGCAGAAGAT CTTAAAGAACTTGAAATTTCAGGGAATTCGATCTGCGTTGATAATCGTTGCTATCCTAAG ATATTTGAACCAΆGACACGATTGGCAGCCCATACTGCCAGGTCAAGAACTCCCCGGTGGT TTGGACATTAGAATAAACATGGACACAGGTTTAAAΆGAGGCAAAACTAΆATGATGΆGAAG AATGTCGGTGATAATGGTAGCCATGAGTTAATTGTATCTTCAGAAGACATGAAAGCATCG CCTGGTGACTATGAATTTTCCAGTGATTTCAΆAGAΆΆTGAGAAΆCATCATAGATTCTAAC CCGACTTTATCTTCACAGGACATTGCCAGATTGGAGGATAGTTTTGATAGAATAATGGAA TTTGCGCATGATTACAAGCΆCGGCTACAAAATTATTACCCATGAΆTTCGCCCTCTTGGCC AACCTTAGTCTCAATGAAAATTTGCCGTTAACATTGAGAGΆGCTCAGTACTΆGAGTCATT ACCAGCTGCTTGAGAAACAATCCTCCTGTAGTCGAGTTCATTΆATGAAAGTTTTCCAAAT TTTAΆAAGCΆAAATCATGGCCGCTCTGTCAAATTTGAATGATTCTAACCACAGATCCTCT AATATCCTAATAAAAAGATACTTGTCCATTTTAAACGAATTACCTGTCACATCCGAAGAT CTTCCTATATACTCTACGGTTGTTTTACAAAATGTATATGAAAGAAACAACAAGGACAAA CAGTTACAAATAAAΆGTCCTGGAGTTGATCAGCΆAAATTTTGAAGGCCGACATGTACGAA AATGACGATACAAATCTAATTTTGTTCAAAAGAΆATGCTGAGAΆTTGGTCGTCAAATCTG CAAGAGTGGGCAAΆCGAGTTCCAAGAGATGGTCCAGAΆCAAAAGTATΆGATGΆACTACAT ACAAGAACGTTTTTTGΆCACCCTTTACAΆCTTGAAGAAAATTTTCAAAAGTGACATCΆCG ATCAΆCAAAGGGTTTTTGAATTGGTTAGCGCAACAATGTAAAGCCAGGCAΆTCTAACTTG GACAATGGGCTCCAAGAGAGAGATACTGAACAAGACTCATTTGΆTAΆGAAΆCTTATCGAC AGCAGACACTTGATCTTTGGCAACCCCATGGCTCATAGAATAAΆAAΆTTTCAGAGATGAA
CTCTGA
Further information on SELl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000005391.
It will be appreciated that, by "SILl", we include fragments or variants (including homologues) thereof having equivalent SILl-like activity. In one embodiment, variants of SILl may or may not include bacterial GrpE type proteins and/or animal (such as mammalian) GrpE-like proteins. Variants of SILl may be a nucleotide exchange factor for an Hsp70 family protein, which nucleotide exchange factor is optionally not an Hsp70 family protein in itself. Suitable variants of SILl may or may not be FESl and/or MGEl. A variant of SILl may or may not be localised to the lumen of the ER (such as SELl itself) to the mitochondria (such as MGEl) or to the cytosol (such as FESl). A variant of SILl may or may not include proteins such as members so of the mammalian GrpE-like protein family, the NEF family or BAG-I (such as described in Hohfeld and Jentsch (1997) EMBO J. 16, 6209), mammalian BiP-associated protein (BAP) (Chung et al (2002) J Biol. Chem. 277, 47557), a human GrpE-like protein (e.g. the protein defined by accession number AAG31605) (Choglay et al (2001) Gene 267, 125), an Λrabidopsis thaliana GrpE-like protein (for example, accession numbers AAK68792 and BAB08589) (Sato et al (1998) DNA Res. 5, 41), a Chlamydia trachomatis Protein grpE (HSP-70 cofactor) (e.g. accession number P36424), a Pongo pygmaeus adenine nucleotide exchange factor (e.g. accession number CAH89792), a Mus musculus mitochondrial GrpE-like 2 protein (e.g. accession number NP_067271), a Mus musculus mitochondrial GrpE-like 1 protein (e.g. accession number NP_077798), a Gallus gallus GrpE protein homolog 2, mitochondrial precursor (Mt-GrpE#2) (e.g. accession number XP_425191), a Gallus gallus BiP-associated protein (e.g. accession number XP_414514), an Haemophilus influenzae 86-028NP GrpE protein (e.g. as defiend by accession number YP_247735) (Harrison et al (2005) J Bacteriol. 187, 4627), an Escherichia coli GrpE heat shock protein (e.g. as defined by accession number NP_417104) (Riley et al (1997) Science 277, 1453), a Streptococcus pneumoniae GrpE heat shock protein (e.g. as defined by accession number AAD23453), a Bacillus subtilis GrpE protein accession number (e.g. as defined by BAA12463) (Mizuno et al (1996) Microbiology (Reading, Engl.) 142, 3103) and/or a Nicotiana tabacum chaperone GrpE type 1 or GrpE type 2 protein (e.g. as defined by accession numbers AAC72386 or AAC72387) (Padidam et al (1999) Plant MoI Biol. 39, 871).
Variants of SILl may have an activity equivalent to SILl, when co-expressed with one or both of JEMl and LHSl, for example in the manner as set out in the present examples. Thus, a host cell of the present invention, when genetically 89
modified to cause simultaneous over-expression of a variant of SILl with one or both of JEMl and LHSl, will provide at least substantially the same increase in the production of a protein product and/or at least substantially the same reduction of fragmentation of a protein product, as is observed in the same host cell when genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl, the increase being compared to the level of production of the same protein product, and/or the level of fragmentation of the same protein product, in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl.
By "substantially the same increase in the production of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the increase in production of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl (the increased being compared to the level of production of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
By "substantially the same reduction of fragmentation of a protein product", we mean at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, substantially 100% or greater than 100% of the reduction of fragmentation of a protein product that is observed when the host cell is genetically modified to cause simultaneous over-expression of SILl with one or both of JEMl and LHSl (the reduction of fragmentation of a protein product being compared to the level of fragmentation of the same protein product in the same host cell that has not been genetically modified to cause overexpression of any of LHSl, JEMl or SILl).
FKB2 is another S. cerevisiae helper protein of interest for the present invention and is also known as FPR2 and FKBPl 3. Fkb2p is a membrane bound peptidyl- prolyl cis-trans isomerase (PPIase) that binds to the drugs FK506 and rapamycin. 89
The expression pattern of Fkb2p suggests possible involvement in ER protein trafficking. A published protein sequence for the protein Fkb2p is as follows:
MMFNIYLFVTFFSTILAGSLSDLEIGIIKRIPVEDCLIKAMPGDKVKVHY TGSLLESGTVFDSSYSRGSPIAFELGVGRVIKGWDQGVAGMCVGEKRKLQ IPSSLAYGERGVPGVIPPSADLVFDVELVDVKSAA*
FKB2 is encoded by a non-essential gene comprising an ORF that is 0.408 kbp in size and is located on chromosome IV. A published nucleotide coding sequence of FKB2 is as follows, 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:
ATGATGTTTAATATTTACCTTTTCGTCACTTTTTTTTCCACCATTCTTGCAGGTTCCCTG TCAGATTTGGAAATCGGTATTATCAAGAGAATACCGGTAGAAGATTGCTTAATTAAGGCA
ATGCCAGGTGATAΆAGTTAAGGTTCATTATACAGGΆTCTTTATTAGAATCGGGAACTGTA
TTTGΆCTCAAGTTATTCAAGAGGCTCTCCTATCGCTTTTGAACTTGGCGTTGGCAGΆGTA
ATTAAAGGTTGGGATCAAGGTGTTGCCGGCATGTGCGTTGGCGAAAAAAGAAAGCTGCAA
ATTCCAAGTTCTTTGGCCTACGGAGAAAGAGGTGTCCCAGGCGTCATTCCTCCAΆGTGCT GATTTGGTGTTTGATGTCGAATTGGTAGACGTGAAATCAGCCGCCTAG
Further information on FKB2 can be obtained from the URL address http ://db .yeastgenome . org/cgi-brn/singlepageformat?sgdid=S 000002927.
It will be appreciated that, by "FKB2", we include fragments or variants thereof having equivalent FKB2-like activity.
SSAl is another S. cerevisiae helper protein of interest for the present invention and is also known as YGlOO. Ssalp is an ATPase that is involved in protein folding and nuclear localization signal (NLS)-directed nuclear transport. It is a member of heat shock protein 70 (HSP70) family. It forms a chaperone complex with Ydjlp and is localized to the nucleus, cytoplasm, and cell wall A published protein sequence for the protein Ssalp is as follows: 6 002289
MSKAVGIDLGTTYSCVAHFANDRVDIIANDQGNRTTPSFVAFTDTERLIGDAAKNQAAMN PSNTVFDAKRLIGRNFNDPEVQADMKHFPFKLIDVDGKPQIQVEFKGETKNFTPEQISSM VLGKMKETAESYLGAKVNDAWTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAΆIA YGLDKKGKEEHVLIFDLGGGTFDVSLLFIEDGIFEVKATAGDTHLGGEDFDNRLVNHFIQ EFKRKNKKDLSTNQRALRRLRTACERAKRTLSSSAQTSVEIDSLFEGIDFYTSITRARFE ELCADLFRSTLDPVEKVLRDAKLDKSQVDEIVLVGGSTRIPKVQKLVTDYFNGKEPNRSI NPDEAVAYGAAVQAAILTGDESSKTQDLLLLDVAPLSLGIETAGGVMTKLIPRNSTISTK KFEIFSTYADNQPGVLIQVFEGERAKTKDNNLLGKFELSGIPPAPRGVPQIEVTFDVDSN GILNVSAVEKGTGKSNKITITNDKGRLSKEDIEKMVAEAEKFKEEDEKESQRIASKNQLE SIAYSLKNTISEAGDKLEQADKDTVTKKAEETISWLDSNTTASKEEFDDKLKELQDIANP IMSKLYQAGGAPGGAΆGGAPGGFPGGAPPAPEAEGPTVEEVD*
SSAl is encoded by a non-essential gene comprising an ORF that is 1.929 kbp in size and is located on chromosome I. A published nucleotide coding sequence of SSAl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGTCAAAAGCTGTCGGTATTGATTTAGGTACAACATACTCGTGTGTTGCTCACTTTGCT AATGATCGTGTGGACATTATTGCCAACGATCAAGGTAACAGAACCACTCCATCTTTTGTC
GCTTTCACTGACACTGAΆAGATTGATTGGTGATGCTGCTAAGAATCAAGCTGCTATGΆAT
CCTTCGAATACCGTTTTCGACGCTAAGCGTTTGATCGGTAGAAACTTCAACGACCCAGAA
GTGCAGGCTGACATGAAGCACTTCCCATTCAAGTTGATCGATGTTGACGGTAAGCCTCAA
ATTCAΆGTTGAATTTAAGGGTGAAACCAAGAΆCTTTACCCCAGAACAAΆTCTCCTCCATG GTCTTGGGTAAGATGAAGGAAACTGCCGAATCTTACTTGGGAGCCAAGGTCAATGACGCT
GTCGTCACTGTCCCAGCTTACTTCAACGATTCTCAAAGACAAGCTACCAAGGATGCTGGT
ACCATTGCTGGTTTGAATGTCTTGCGTATTATTAACGAACCTACCGCCGCTGCCΆTTGCT
TACGGTTTGGACAAGAAGGGTAAGGAAGAΆCACGTCTTGATTTTCGACTTGGGTGGTGGT
ACTTTCGATGTCTCTTTGTTGTTCATTGAΆGACGGTATCTTTGΆAGTTAAGGCCACCGCT GGTGACACCCATTTGGGTGGTGAAGATTTTGACAACAGATTGGTCAACCACTTCATCCAA
GAATTCAΆGAGAAAGAACAAGAΆGGΆCTTGTCTACCAACCAAAGAGCTTTGAGAAGATTA
AGAACCGCTTGTGAAAGAGCCAAGAGAACTTTGTCTTCCTCCGCTCAAACTTCCGTTGAA ATTGACTCTTTGTTCGΆAGGTATCGATTTCTACACTTCCATCACCAGAGCCAGATTCGAA GAATTGTGTGCTGACTTGTTCAGATCTACTTTGGACCCAGTTGAAAAGGTCTTGAGAGAT GCTAAATTGGACAAATCTCAAGTCGATGAAATTGTCTTGGTCGGTGGTTCTACCAGAATT CCAAAGGTCCAAAAΆTTGGTCACTGΆCTACTTCAΆCGGTAAGGAACCAAΆCAGATCTATC AACCCAGATGAAGCTGTTGCTTACGGTGCTGCTGTTCAAGCTGCTATTTTGACTGGTGAC GAATCTTCCAAGACTCAAGATCTATTGTTGTTGGATGTCGCTCCΆTTATCCTTGGGTATT GAAACTGCTGGTGGTGTCATGACCAAGTTGATTCCAAGAAACTCTACCATTTCAACAAAG AAGTTCGAGATCTTTTCCACTTATGCTGATAACCAACCAGGTGTCTTGATTCAAGTCTTT GAAGGTGAAAGAGCCAAGΆCTAAGGACAACAACTTGTTGGGTAAGTTCGAATTGAGTGGT ATTCCACCAGCTCCAΆGAGGTGTCCCACAAATTGAAGTCACTTTCGATGTCGACTCTAAC GGTATTTTGAATGTTTCCGCCGTCGAAAAGGGTACTGGTAAGTCTAACAAGATCACTATT ACCAACGACAAGGGTAGATTGTCCAAGGAAGATATCGAAAAGATGGTTGCTGAAGCCGAA AAATTCAAGGAAGAAGATGAAAAGGAATCTCAAAGAATTGCTTCCAΆGAΆCCAATTGGAA TCCATTGCTTACTCTTTGAAGAACACCATTTCTGAAGCTGGTGACAAATTGGAACAAGCT GACAAGGACACCGTCACCAΆGAAGGCTGAAGAGACTATTTCTTGGTTAGACAGCAACACC ACTGCCAGCAAGGAAGAATTCGATGACAAGTTGAAGGAGTTGCAAGACATTGCCAACCCA ATCATGTCTAAGTTGTACCAAGCTGGTGGTGCTCCAGGTGGCGCTGCAGGTGGTGCTCCA GGCGGTTTCCCAGGTGGTGCTCCTCCAGCTCCAGAGGCTGAAGGTCCAACCGTTGAAGAA GTTGATTAA
Further information on SSAl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000004.
It will be appreciated that, by "SSAl", we include fragments or variants thereof having equivalent SSAl -like activity.
SSA2 is another S. cerevisiae helper protein of interest for the present invention.
Ssa2p is an ATP binding protein that is involved in protein folding and vacuolar import of proteins; member of heat shock protein 70 (HSP70) family. It is associated with the chaperoriin-containing T-complex. It is present in the cytoplasm, vacuolar membrane and cell wall. A published protein sequence for the protein Ssa2p is as follows:
MSKAVGIDLGTTYSCVAHFSNDRVDIIANDQGNRTTPSFVGFTDTERLIGDAAKNQAAMN
PANTVFDAKRLIGRNFNDPEVQGDMKHFPFKLIDVDGKPQIQVEFKGETKNFTPEQISSM VLGKMKETAESYLGAKVNDAVVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIA
YGLDKKGKEEHVLIFDLGGGTFDVSLLSIEDGIFEVKATAGDTHLGGEDFDNRLVNHFIQ EFKRKNKKDLSTNQRALRRLRTACERAKRTLSSSAQTSVEIDSLFEGIDFYTSITRARFE ELCADLFRSTLDPVEKVLRDAKLDKSQVDEIVLVGGSTRIPKVQKLVTDYFNGKEPNRSI NPDEAVAYGAAVQAAILTGDESSKTQDLLLLDVAPLSLGIETAGGVMTKLIPRNSTIPTK KSEVFSTYADNQPGVLIQVFEGERAKTKDNNLLGKFELSGIPPAPRGVPQIEVTFDVDSN GILNVSAVEKGTGKSNKITITNDKGRLSKEDIEKMVAEΆEKFKEEDEKESQRIASKNQLE SIAYSLKNTISEAGDKLEQADKDAVTKKAEETIAWLDSNTTATKEEFDDQLKELQEVANP IMSKLYQAGGAPEGAAPGGFPGGAPPAPEAEGPTVEEVD *
SSA2 is encoded by a non-essential gene comprising an ORF that is 1.920 kbp in size and is located on chromosome XII. A published nucleotide coding sequence of SSA2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGTCTAAAGCTGTCGGTATTGATTTAGGTACTACCTACTCCTGTGTTGCTCACTTCTCT AATGATCGTGTTGACATTATTGCCAACGACCAAGGTAACAGAACCACTCCATCTTTCGTT GGTTTCACTGATACTGAAAGATTGATTGGTGACGCTGCTAAGAACCAAGCTGCTATGAAC CCAGCTAACACTGTTTTCGACGCTAAGCGTTTGATCGGTAGAAACTTCAATGACCCAGAA GTCCAAGGTGATATGAAGCACTTCCCATTCAAGTTGATCGATGTTGACGGTAAGCCACAA ATTCAAGTTGAATTTAAGGGTGAAACCAAGAACTTTACCCCAGAACAAATCTCCTCCATG GTCTTGGGTAAGATGAAGGAAACTGCCGAATCTTACTTGGGTGCCAAGGTCAATGACGCT GTCGTCACTGTCCCAGCTTACTTCAACGATTCTCAAAGACAAGCTACCAAGGATGCTGGT ACCATTGCTGGTTTGAATGTCTTGCGTATTATTAACGAACCTACCGCCGCTGCCATTGCT TACGGTTTGGACAAGAAGGGTAAGGAAGAACACGTCTTGATTTTCGACTTGGGTGGTGGT ACTTTCGATGTCTCTTTGTTGTCCATTGAAGACGGTATCTTTGAAGTTAAGGCCACCGCT GGTGACACCCATTTGGGTGGTGAAGATTTTGACAACAGATTGGTCAACCACTTCATCCAA GAATTCΆAGAGAAAGAACAΆGAAGGACTTGTCTACCAACCAAAGAGCTTTGAGAAGATTA AGAACTGCTTGTGAAAGAGCCAAGAGAACTTTGTCTTCCTCCGCTCAAACTTCCGTTGAA ATTGACTCTTTGTTCGAΆGGTATCGATTTCTACACTTCCATCACCAGAGCCAGATTCGAA GAATTGTGTGCTGACTTGTTCAGATCTACTTTGGACCCAGTTGAAAAGGTCTTGAGAGAT GCTAAATTGGATAAATCTCAAGTCGATGAAATTGTCTTGGTCGGTGGTTCTACCAGAATT CCAAAGGTCCAAAAATTGGTCACTGACTACTTCAACGGTAAGGAACCAAACAGATCTATC AACCCAGATGAAGCTGTTGCTTACGGTGCTGCTGTTCAAGCTGCTATTTTGACTGGTGAC GAATCTTCCAAGACTCAAGATCTATTGTTGTTGGATGTCGCTCCATTATCCTTGGGTATT GAAACTGCTGGTGGTGTCATGACCΆAGTTGATTCCAAGAAACTCTACCATTCCAACTAAG AAATCCGAAGTTTTCTCTACTTATGCTGACAACCAACCAGGTGTCTTGATTCAAGTCTTT GAAGGTGAAAGAGCCAAGACTAAGGACAΆCAACTTGTTGGGTAAGTTCGAΆTTGAGTGGT ATTCCACCAGCTCCAAGAGGTGTCCCACAΆATTGAAGTCACTTTCGATGTCGACTCTAAC GGTATTTTGAATGTTTCCGCCGTCGAAAAGGGTACTGGTAAGTCTAACAAGATCACTATT ACCAACGACAAGGGTAGATTGTCCAAGGAAGATATCGAAAAGATGGTTGCTGAAGCCGAA AAATTCAAGGAAGAAGATGAAAAGGAATCTCAAAGAATTGCTTCCAAGAACCAATTGGAA TCCATTGCTTACTCTTTGAAGAACACCATTTCTGAAGCTGGTGACAAGCTAGAGCAAGCT GACΆΆGGACGCTGTCACTAAGAAGGCTGAAGAAACTATTGCTTGGTTAGACAGCAACACC ACTGCTACCAAGGAAGAATTCGATGACCAATTGAAGGAATTGCAAGAGGTTGCCAACCCA ATCATGTCTAAATTGTACCAAGCTGGTGGTGCTCCAGAAGGCGCAGCTCCAGGTGGTTTC
CCAGGTGGTGCTCCTCCAGCTCCAGAAGCTGAAGGTCCAACTGTCGAAGAAGTTGATTAA
Further information on SSA2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000003947.
It will be appreciated that, by "SSA2", we include fragments or variants thereof having equivalent SSA2-like activity.
SS A3 is another S. cerevisiae helper protein of interest for the present invention, which is also known as HSP70. Ssa3p is an ATPase involved in protein folding and the response to stress. It plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation and is a member of the heat shock protein 70 (HSP70) family. S S A3 is localized to the cytoplasm. A published protein sequence for the protein Ssa3p is as follows:
MSRAVGIDLGTTYSCVAHFSNDRVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQAAIN PHNTVFDAKRLIGRKFDDPEVTTDAKHFPFKVISRDGKPWQVEYKGETKTFTPEEISSM VLSKMKETAENYLGTTVNDAWTVPAYFNDSQRQATKDAGTIAGMNVLRIINEPTAAAIA YGLDKKGRAEHNVLIFDLGGGTFDVSLLSIDEGVFEVKATAGDTHLGGEDFDNRLVNHLA TEFKRKTKKDISNNQRSLRRLRTAAERAKRΆLSSSSQTSIEIDSLFEGMDFYTSLTRARF EELCADLFRSTLEPVEKVLKDSKLDKSQIDEIVLVGGSTRIPKIQKLVSDFFNGKEPNRS INPDEAVAYGAAVQAAILTGDQSTKTQDLLLLDVAPLSLGIETAGGIMTKLIPRNSTIPT KKSETFSTYADNQPGVLIQVFEGERTRTKDNNLLGKFELSGIPPAPRGVPQIDVTFDIDA NGILNVSALEKGTGKSNKΪTITNDKGRLSKDDIDRMVSEAEKYRADDEREAERVQAKNQL ESYAFTLKNTINEASFKEKVGEDDAKRLETASQETIDWLDASQAASTDEYKDRQKELEGI ANPIMTKFYGAGAGAGPGAGESGGFPGSMPNSGATGGGEDTGPTVEEVD*
SSA3 is encoded by a non-essential gene comprising an ORF that is 1.950 kbp in size and is located on chromosome II. A published nucleotide coding sequence of SSA3 is as follows, 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:
ATGTCTAGAGCAGTTGGTATTGATTTGGGAACAACTTACTCGTGTGTTGCTCATTTTTCC B2006/002289
AATGATAGGGTAGAGATAATTGCΆAATGATCAΆGGTAATAGGACCACTCCATCGTATGTG GCTTTCACAGACACCGAAΆGATTAATTGGTGACGCCGCCAAAAATCAΆGCTGCAATCAAT CCTCATAATACAGTTTTTGATGCAAAGCGGTTAATTGGTCGTAAATTTGATGATCCTGAA GTGACGACAGATGCCAAGCACTTCCCTTTCAΆAGTTATATCCAGAGATGGTAAΆCCTGTA GTGCAAGTAGAATATAAGGGTGAAACGAAAACATTTACGCCTGAGGAAATTTCTTCCATG GTTTTAAGCAAAATGAAGGAAACTGCTGAGAΆCTATTTGGGAACTACGGTCAΆTGATGCT GTTGTAACTGTTCCTGCATATTTCAATGATTCTCAAAGACAΆGCCACTAΆGGATGCAGGA ACTATTGCAGGGATGAACGTTTTACGTATTATCAATGAACCCACTGCAGCAGCAATTGCT TATGGCTTGGATAAGAAAGGCAGGGCTGAGCACAATGTCCTGATTTTTGATTTGGGTGGT GGTACTTTTGACGTCTCTTTACTTTCAATTGATGAGGGTGTTTTTGAGGTTAAGGCTACC GCAGGAGACACTCATTTAGGTGGTGAAGATTTTGATAATAGGTTGGTGAACCATTTAGCC ACTGAΆTTCAAAAGGAAAACGAAAAAGGACATCTCTAATAATCAAΆGATCGTTAAGAAGΆ TTGAGAACTGCGGCAGAAΆGAGCTAΆGAGAGCGCTTTCTTCCTCATCTCAAACCTCGATC GAGATCGATTCTTTATTTGAAGGTATGGATTTCTACACTTCGTTAACAAGGGCAAGGTTT GAAGAGCTATGTGCTGATTTATTCAGATCCACATTGGAACCAGTAGAAAAGGTTCTTAAA GATTCGAAGCTGGACAAGTCCCAAATTGATGAGΆTTGTGTTAGTCGGTGGATCTACCAGA ATCCCAAAGATTCAGAAATTAGTTTCTGACTTCTTCAATGGCAAAGAGCCTAATCGTTCT ATCAACCCGGATGAGGCTGTTGCTTATGGTGCAGCCGTTCAAGCTGCCATTTTAACCGGC GATCAATCAACAAAGACACAAGATTTACTATTATTGGATGTTGCGCCATTGTCCCTAGGA ATTGAΆACTGCAGGCGGCATAΆTGACTAAGCTAATTCCTAGAAΆCTCAACGATTCCAΆCA AAGAAATCGGAAACCTTCTCTACCTΆTGCAGATAATCAACCTGGTGTTTTAATTCAAGTC TTTGAAGGTGAAAGAACAAGAACAAAGGATAATAACTTACTTGGTAAATTCGAATTAAGT GGCATTCCGCCTGCTCCCAGAGGTGTGCCTCAAATTGATGTTACCTTTGATATCGACGCT AATGGTATTCTTAΆTGTGTCTGCTTTGGAΆAAGGGTACTGGTAAGAGTAACAAAATCACG ATCACTAACGATAAAGGTAGGCTCTCGAAGGATGATATTGATAGGATGGTTTCTGAAGCT GAAAAATATAGGGCTGΆCGATGAAAGGGAGGCAGAACGAGTTCAGGCTAAGAATCAGCTT GΆΆTCGTATGCATTTACTTTGAΆGAATACCATAAACGAAGCAAGTTTCAAAGAGAAAGTA GGTGAAGATGATGCAAAGAGATTAGAAACAGCGTCTCAGGAAACCATTGACTGGTTAGAT GCATCGCAGGCAGCCTCTACGGACGAATATAAGGATAGACAAAAGGAGTTGGAAGGCATT GCCAATCCAATAATGACGAAATTTTACGGTGCTGGTGCCGGCGCAGGTCCTGGAGCGGGG GAΆTCCGGTGGATTCCCCGGATCCATGCCCAACTCGGGTGCTACGGGAGGTGGAGAAGAT ACAGGTCCAACAGTGGAAGAGGTTGATTGA
Further information on SSA3 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000171.
It will be appreciated that, by "SSA3", we include fragments or variants thereof having equivalent SSA3-like activity. SSA4 is another S. cerevisiae helper protein of interest for the present invention. Ssa4p is a heat shock protein that is highly induced upon stress. It plays a role in SRP-dependent cotranslational protein-membrane targeting and translocation; member of the HSP70 family. It is a cytoplasmic protein that concentrates in nuclei upon starvation. A published protein sequence for the protein Ssa4p is as follows:
MSKΆVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSYVAFTDTERLIGDAΆKNQAAMN PHNTVFDAKRLIGRKFDDPEVTNDAKHYPFKVIDKGGKPWQVEYKGETKTFTPEEISSM ILTKMKETAENFLGTEVKDAVVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIA YGLDKKSQKEHNVLIFDLGGGTFDVSLLSIDEGVFEVKATAGDTHLGGEDFDSRLVNFLA EEFKRKNKKDLTTNQRSLRRLRTΆAERAKRTLSSSAQTSIEIDSLFEGIDFYTSITRARF EELCADLFRSTLEPVEKVLADSKLDKSQIDEIVLVGGSTRIPKVQKLVSDFFNGKEPNRS INPDEAVAYGAAVQAAILTGDQSSTTQDLLLLDVAPLSLGIETAGGIMTKLIPRNSTIPT KKSEVFSTYADNQPGVLIQVFEGERTRTKDNNLLGKFELSGIPPAPRGVPQIEVTFDIDA NGILNVSAVEKGTGKSNKITITNDKGRLSKEDIDKMVAEAEKFKAEDEQEAQRVQΆKNQL ESYAFTLKNSVSENNFKEKVGEEDARKLEAAAQDAINWLDASQAASTEEYKERQKELEGV ANPIMSKFYGAAGGAPGAGPVPGAGAGPTGAPDNGPTVEEVD*
SSA4 is encoded by anon-essential gene comprising an ORF that is 1.929 kbp in size and is located onchromosomeV. Apublishednucleotide coding sequence of SSA4 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
ATGTCAAAAGCTGTTGGTATTGATTTAGGTACAACCTATTCATGTGTTGCTCATTTTGCA AACGATAGGGTTGAAATTATCGCTAACGATCAAGGTAATAGAACGACGCCTTCTTATGTG GCTTTTACTGACACAGAAAGGCTAATTGGTGACGCTGCGAAGAATCAAGCTGCGATGAAC CCACATAATACAGTATTCGATGCTAAGCGTCTGATCGGACGTAAATTCGATGATCCAGAA GTGACGAACGATGCTAAGCATTACCCATTCAAAGTGATTGACAAGGGAGGTAAACCGGTA GTGCAAGTGGAATATAAAGGCGAGACAAΆGACATTTACTCCAGAAGAAΆTTTCCTCAATG ATCTTGACAAAGATGAAGGAGACTGCTGAGAACTTTTTAGGAACAGAAGTGAAAGATGCT GTAGTAACGGTTCCAGCCTATTTCAACGATTCACAAAGGCAΆGCAACAΆAAGATGCCGGT ACAATCGCGGGCTTGAACGTTCTTCGTATCATTAATGAACCTACAGCTGCCGCTATTGCG TATGGGCTGGACAAGAAATCGCAGAAGGAGCACAACGTCTTGΆTCTTTGATTTAGGTGGT GGTACTTTTGATGTCTCTCTGCTATCCATAGATGAAGGTGTCTTTGAGGTTAAGGCTACT
GCTGGTGACΆCTCACTTGGGTGGTGAΆGATTTCGATAGTAGGCTGGTTAACTTTCTAGCC GAGGAGTTCAAAAGAAAAAATAAAAAGGATCTAΆCAACTAACCAAAGGTCCCTAAGGAGG TTAAGGACCGCCGCTGAΆAGGGCCAAGAGAACTCTGTCTTCGTCTGCTCAGACATCTATA GAAATAGATTCATTATTTGAGGGTATCGATTTCTATACTTCCATTACAAGGGCAAGATTT GAAGAATTATGTGCTGATTTGTTTAGATCTACATTGGAGCCAGTGGAAAAAGTTTTGGCT GATTCAAAATTAGATAAGTCACAAATTGATGAAATTGTACTTGTTGGTGGTTCAACAAGA ATTCCAAAAGTACAAAAACTGGTTTCTGATTTTTTCAATGGTAAAGAACCAAACCGTTCG ATTAACCCTGATGAGGCCGTCGCTTATGGTGCTGCCGTACAGGCTGCCATCTTAΆCGGGT GACCAGTCGTCGACGACCCAAGATTTACTGTTGCTGGATGTTGCACCATTATCTCTAGGT ATTGAAACTGCAGGTGGTATTATGACAΆAGTTGATCCCAAGAAATTCGACTATCCCAACA AAAAΆΆTCGGAAGTGTTTTCCACCTACGCTGACAACCAACCTGGTGTGTTGATACAAGTT TTTGAGGGTGAAAGGACAAGGACAAAAGACAACAATCTACTGGGTAAATTTGAGTTGAGC GGTATTCCACCCGCTCCAAGAGGCGTACCACAAATTGAAGTTACATTTGATATCGATGCA AATGGTATTCTGAACGTATCTGCCGTTGAAAAAGGTACTGGTAAATCTAACAAGATTACA ATTACTAACGATAAGGGAAGATTATCGAAGGAAGATATCGATAAAATGGTTGCTGAGGCA GAAAAGTTCAAGGCCGAAGATGAACAAGAAGCTCAACGTGTTCAAGCTAAGAATCAGCTA GAATCGTACGCGTTTACTTTGAAAAATTCTGTGAGCGAAAATAACTTCAAGGAGAAGGTG GGTGAAGAGGATGCCAGGAAATTGGAAGCCGCCGCCCAAGATGCTATAAATTGGTTAGAT GCTTCGCAAGCGGCCTCCACCGAGGAATACAAGGAAAGGCAAAAGGAACTAGAAGGTGTT GCAAACCCCATTATGAGTAΆATTTTACGGAGCTGCAGGTGGTGCCCCAGGAGCAGGCCCA GTTCCGGGTGCTGGAGCAGGCCCCACTGGAGCACCAGACAACGGCCCAACGGTTGAAGAG GTTGATTAG
Further information on SSA4 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000905.
It will be appreciated that, by "SSA4", we include fragments or variants thereof having equivalent SSA4-like activity.
SSEl is another S. cerevisiae helper protein of interest for the present invention and is also known as LP G3 and MSI3. S se Ip is an ATPase that is a component of the heat shock protein Hsp90 chaperone complex. It binds unfolded proteins and is a member of the heat shock protein 70 (HSP70) family. It is localized to the cytoplasm. A published protein sequence for the protein Sselp is as follows:
MS T PFGLDLGNNNSVLAVARNRGI DI WNEVSNRSTP S WGFGPKNRYLGETGKNKQT SN IKNTVANLKRIIGLDYHHPDFEQESKHFTSKLVELDDKKTGAEVRFAGEKHVFSATQLAA MFIDKVKDTVKQDTKANITDVCIAVPPWYTEEQRYNIADAARIAGLNPVRIVNDVTAAGV SYGIFKTDLPEGEEKPRIVAFVDIGHSSYTCSIMAFKKGQLKVLGTACDKHFGGRDFDLA ITEHFADEFKTKYKIDIRENPKAYNRILTAAEKLKKVLSANTNAPFSVESVMNDVDVSSQ LSREELEELVKPLLERVTEPVTKALAQAKLSAEEVDFVEIIGGTTRIPTLKQSISEAFGK PLSTTLNQDEAIAKGAAFICAIHSPTLRVRPFKFEDIHPYSVSYSWDKQVEDEDHMEVFP AGSSFPSTKLITLNRTGDFSMAASYTDITQLPPNTPEQIANWEITGVQLPEGQDSVPVKL KLRCDPSGLHTIEEAYTIEDIEVEEPIPLPEDAPEDAEQEFKKVTKTVKKDDLTIVAHTF GLDAKKLNELIEKENEMLAQDKLVAETEDRKNTLEEYIYTLRGKLEEEYAPFASDAEKTK LQGMLNKAEEWLYDEGFDSIKAKYIAKYEELASLGNIIRGRYLAKEEEKKQAIRSKQEAS QMAAMAEKLAAQRKAEAEKKEEKKDTEGDVDMD*
SSEl is encoded by a non-essential gene comprising an ORF that is 2.082 kbp in size and is located on chromosome XVI. A published nucleotide coding sequence of SSEl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identicalproteinproduct:
ATGAGTACTCCATTTGGTTTAGATTTAGGTAACAATAACTCTGTCCTTGCCGTTGCTAGA AACAGAGGTATCGACATTGTCGTTAATGAAGTCTCTAACCGTTCCACCCCATCTGTTGTT GGTTTTGGTCCAAAGAACAGATACTTGGGTGAAΆCTGGTAAGAACAAGCAGACTTCCAAC ATCAAGAACACTGTCGCCAΆCTTGAAAAGAATTATTGGTTTGGATTACCACCATCCAGAT TTCGAGCAAGAATCTAAGCACTTCACCTCTAAGTTGGTTGAATTGGATGACAΆGAAGACT GGTGCCGAAGTTAGATTCGCTGGTGAGAAACATGTTTTTTCAGCTACTCAACTAGCTGCC ATGTTCATCGACAAAGTCAAGGACACCGTCAAGCAGGACACAAAGGCAAATATTACCGAT GTTTGTATTGCTGTCCCACCTTGGTACACCGAAGAACAACGTTACAACATTGCTGATGCT GCTAGAATTGCTGGTTTGAACCCTGTTAGAATTGTCAACGACGTTACTGCTGCCGGTGTT TCTTACGGTATCTTCAAGACTGATTTGCCTGAAGGCGAAGAAAAGCCAAGΆATTGTTGCC TTTGTTGATATTGGTCACTCTTCCTACACCTGTTCTATCATGGCCTTCAAGAAGGGTCAA TTGAAAGTCTTAGGAACTGCCTGCGACAAGCATTTTGGTGGTAGGGACTTCGATTTGGCT ATAACAGAACATTTCGCCGATGΆGTTCAAAΆCTAAATACAAGATTGACATCAGAGAΆAAT CCAAAGGCTTACAACAGAATTCTAACTGCTGCTGAAAAGTTGAAGAAAGTTTTGTCTGCT AATACTAATGCCCCATTCTCTGTTGAATCCGTCATGAACGACGTTGATGTTTCCTCTCAA TTATCTCGTGAAGAATTAGAAGAATTGGTCAAGCCATTGTTGGAACGTGTTACTGAACCA GTTACCAAAGCTTTAGCTCAAGCCAAATTATCTGCTGAAGAAGTTGATTTTGTTGAAATT ATTGGTGGTACTACTCGTATCCCAACATTGAAACAATCCATTTCTGAΆGCCTTCGGCAAG CCATTGTCCACCACTTTGAACCAAGATGAAGCCATCGCCAAGGGTGCCGCCTTTATTTGC GCCATTCACTCTCCAACTCTAAGAGTTAGACCATTCAAGTTTGAGGATATCCATCCTTAC TCTGTCTCTTACTCTTGGGACAAGCAAGTTGAGGACGAAGACCACATGGAAGTTTTCCCA
GCTGGTTCATCCTTCCCATCTACTAAATTGATCACTTTGAACCGTACGGGTGACTTTTCA
ATGGCTGCTAGCTACACTGACATCACACAGTTACCACCAAACACTCCAGAACAAATCGCT
AΆCTGGGAGATCACTGGTGTTCAATTACCAGAAGGTCAAGACTCTGTTCCTGTTAAGTTA AAGTTGAGATGCGACCCCTCTGGTTTACACACAATTGAAGAGGCTTACACTATTGAAGAT
ATTGAAGTTGAAGAACCTATTCCATTACCAGAAGATGCTCCAGAAGATGCTGAGCAAGAA
TTTAAGAAGGTTACTAAAACTGTAAAGAAGGATGΆCTTΆACCATCGTTGCACACACCTTT
GGCCTAGACGCTAΆAAAGTTGAATGAATTAATTGAAAΆAGAAAATGAΆATGCTTGCTCAA
GATAAGCTAGTTGCTGAGACAGAAGACCGTAAGAACACTCTTGAAGAGTACATCTACACA TTGCGTGGTAAGTTGGAAGAAGAGTATGCTCCATTTGCTTCCGATGCTGAAAAGACGAAG
TTACAAGGTATGTTAAACAAGGCCGAAGAGTGGTTATACGATGAAGG1TTTCGATTCCATC
AAAGCTAAGTACATTGCCAAATACGAAGAATTGGCTTCTCTAGGTAACATTATTAGAGGT AGATACTTGGCTAΆΆGAΆGAΆGAAAAGAAGCAΆGCTATAAGATCTAAGCAΆGAAGCATCC CAAATGGCTGCTATGGCTGAAAAGTTGGCTGCTCAAAGAAAGGCAGAAGCTGAAΆAGAAG GAAGAAAAGAAGGACACTGAAGGTGATGTTGACATGGACTAA
Further information on SSEl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageforniat?sgdid=S000006027.
It will be appreciated that, by "SSEl", we include fragments or variants thereof having equivalent S SEl -like activity.
SSE2 is another S. cerevisiae helper protein of interest for the present invention. Sse2p is a member of the heat shock protein 70 (HSP70) family. It may be involved in protein folding and is localised to the cytoplasm. It is highly homologous to the heat shock protein Sselp. A published protein sequence for the protein Sse2p is as follows:
MSTPFGLDLGNNNSVLAVARNRGIDVWNEVSNRSTPSLVGFGPRNRYLGESGKTKQTSN VKNTVENLKRIIGLKFKDPEFDIENKFFTSKLVQLKNGKVGVEVEFGGKTHVFSATQLTA
MFIDKVKHTVQEETKSSITDVCLAVPVWYSEEQRYNIADAARIAGLNPVRIVNDVTAAAV
SYGVFKNDLPGPEEKPRIIGLVDIGHΞTYTCSIMAFRKGEMKVLGTAYDKHFGGRDFDRA
ITEHFADQFKDKYKIDIRKNPKAYNRILIAAEKLKKVLSANTTAPFSVESVMDDIDVSSQ
LSREELEELVEPLLKRVTYPITNALAQAKLTVNDIDFVEIIGGTTRIPVLKKSISDVFGK PLSSTLNQDEAVAKGAAFICAIHSPTLRVRPFKFEDIDPYSVSYTWDKQVDDEDRLEVFP
ANSSYPSTKLITLHRTGDFSMKAVYTHPSKLPKGTSTTIAKWSFTGVKVPKDQDFIPVKV
KLRCDPSGLHIIENAYTTEDITVQEPVPLPEDAPEDAEPQFKEVTKTIKKDVLGMTAKTF ALNPVELNDLIEKENELRNQDKLVAETEDRKNALEEYIYTLRAKLDDEYSDFASDAEKEK LKNMLATTENWLYGDGDDSTKAKYIAKYEELASLGNIIRGRYLAKEΞEKRQALRANQETS KMNDIAEKLAEQRRARAASDDSDDNNDENMDLD*
SSE2 is encoded by a non-essential gene comprising an ORP that is 2.082 kbp in size and is located on chromosome II. A published nucleotide coding sequence of SSE2 is as follows, 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:
ATGAGCACTCCATTTGGCTTAGATTTAGGTAACAATAACTCAGTACTAGCAGTTGCCAGA AATAGGGGTATTGATGTCGTTGTCAATGAAGTTTCTAATAGGTCTACACCATCCTTGGTC GGCTTTGGCCCCAGAAATAGGTACTTAGGTGAΆTCTGGTAAAACTAAGCAAACATCGAAT GTTAAAAACACTGTGGAAAACTTGΆΆAAGAATCATTGGACTAAAGTTCAAAGACCCTGAA TTTGATATCGAGAATAAGTTCTTCACTTCGAAATTGGTACAGCTAAAAAATGGTAAAGTT GGTGTGGAAGTGGAGTTCGGCGGTAAAACACACGTATTTTCAGCTACTCAΆCTGACTGCT ATGTTCATTGATAAGGTGAAGCACACCGTTCAΆGAGGAΆACGAAGTCATCAATTACCGAT GTCTGCCTCGCAGTTCCTGTATGGTATTCGGAAGAACAACGTTATAACATAGCCGATGCT GCCAGAATTGCAGGATTAAATCCTGTAAGGATTGTCAΆCGATGTGACTGCAGCCGCCGTT TCGTACGGCGTCTTCAAGAATGATCTGCCAGGTCCTGAAGAAAAGCCAAGAATCATTGGC TTAGTGGACATTGGGCATTCTACCTACACCTGTTCTATTATGGCTTTCCGCAAAGGCGAA ATGAAAGTATTAGGTACTGCTTATGACAAGCACTTTGGTGGTAGAGATTTCGATCGCGCA ATCACAGAACATTTTGCTGATCAGTTTAAGGACAAGTACAAGATTGACATTAGGAAAAAT CCGAAAGCTTATAACAGAATTTTAATCGCTGCTGAAAAATTAAAAAAAGTGCTTTCTGCG AACACTACTGCCCCCTTCTCCGTTGAATCTGTTATGGATGATATCGACGTTTCCTCTCAA TTGAGCCGTGAAGAGCTGGAAGAATTAGTAGAGCCCTTGTTGAAGCGTGTGACGTATCCA ATCACCAATGCATTGGCTCAAGCTAAATTAACTGTCΆATGATATTGACTTCGTAGAAATA ATTGGTGGTACAACCCGTATCCCAGTTTTAAAGAAGTCAATTTCTGATGTTTTTGGAAΆA CCTTTGTCATCTACTTTAAATCAAGACGAAGCTGTGGCCAAGGGGGCCGCTTTCATATGT GCCATTCACTCTCCAACTTTAAGGGTCAGGCCGTTTAAATTTGAAGATATTGATCCGTAT TCAGTGTCATACACTTGGGATAAGCAGGTCGATGACGAAGACCGTTTGGAΆGTATTCCCT GCTAATTCATCATATCCATCAACTAAACTΆATTACTTTACATCGTACTGGAGΆTTTCAGC ATGAAAGCGGTGTACACTCATCCTTCGAAACTGCCAAAAGGTACTTCCACCACTATTGCA AAATGGAGCTTCACTGGGGTCAAGGTTCCTAAAGATCAAGATTTTATTCCTGTAAAGGTC AAGTTAAGATGCGATCCTTCCGGCTTGCATATTATCGAGAACGCTTACACAACGGAAGAT ATTACGGTTCAAGAGCCAGTGCCTTTACCGGAAGACGCACCAGAΆGATGCCGAGCCCCAG TTTAAAGAAGTTACTAAAACAATTAAGAAAGATGTGCTAGGTATGACTGCAAAAACATTC
GCGCTAAACCCGGTTGAGTTGAACGATCTAATTGAAAAAGAGAATGAATTAAGAAACCAG GATAAGTTAGTTGCCGAΆACCGAGGATCGCAAAAATGCCCTTGAAGAGTATΆTTTATACC CTTCGTGCCAΆACTCGATGATGAATACTCCGATTTTGCGTCTGACGCAGAAAAAGAAAAG CTAAAAAACATGTTAGCCACTACTGAAAΆTTGGTTATATGGTGATGGTGACGATTCTACC AAGGCAAAATACATTGCTAAATATGAGGAGCTGGCATCGTTGGGGAΆTATTΆTTAGAGGT AGATATTTAGCAAAGGAGGAAGAAAAAAGACAAGCACTCAGAGCGAATCAAGAAACTTCT AAAATGAATGATATTGCTGAAAAATTGGCTGAGCAAAGAAGGGCACGCGCTGCAAGTGAT GATAGCGATGACAACAATGATGAAAACATGGACCTTGATTAA
Further information on SSE2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000373.
It will be appreciated that, by "SSE2", we include fragments or variants thereof having equivalent SSE2-like activity.
SSBl is another S. cerevisiae helper protein of interest for the present invention and is also known as YGlOl. Ssblp is a cytoplasmic ATPase that is a ribosome- associated molecular chaperone. It may be involved in the folding of newly- synthesized polypeptide chains and is a member of the heat shock protein 70 (HSP70) family. It interacts with the phosphatase subunit Reglp. A published protein sequence for the protein Ssblp is as follows:
MAEGVFQGAIGIDLGTTYSCVATYESSVEIIANEQGNRVTPSFVAFTPEERLIGDAAKNQ AALNPRNTVFDAKRLIGRRFDDESVQKDMKTWPFKVIDVDGNPVIEVQYLEETKTFSPQE ISAMVLTKMKEIAEAKIGKKVEKAVITVPAYFNDAQRQATKDAGAISGLNVLRIINEPTA AAIAYGLGAGKSEKERHVLIFDLGGGTFDVSLLHIAGGVYTVKSTSGNTHLGGQDFDTNL LEHFKAEFKKKTGLDISDDARALRRLRTAAERAKRTLSSVTQTTVEVDSLFDGEDFESSL TRARFEDLNAALFKSTLEPVEQVLKDAKISKSQIDEWLVGGSTRIPKVQKLLSDFFDGK QLEKSINPDEAVAYGAAVQGAILTGQSTSDETKDLLLLDVAPLSLGVGMQGDMFGiyVPR NTTVPTIKRRTFTTCADNQTTVQFPVYQGERVNCKENTLLGEFDLKNIPMMPAGEPVLEA IFEVDANGILKVTAVEKSTGKSSNITISNAVGRLSSEEIEKMVNQAEEFKAADEAFAKKH EARQRLESYVASIEQTVTDPVLSSKLKRGSKSKIEAALSDALAALQIEDPSADELRKAEV GLKRWTKAMSSR*
SSBl is encoded by a non-essential gene comprising an ORF that is 1.842 kbp in size and is located on chromosome IY. A published nucleotide coding sequence of SSBl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGGCTGAAGGTGTTTTCCAAGGTGCTATCGGTATCGATTTAGGTACAACCTACTCTTGT GTTGCTACTTACGAATCCTCCGTTGAAATTATTGCCAACGAACAAGGTAACAGAGTCACC CCATCTTTCGTTGCTTTCACTCCAGAAGAΆAGATTGATTGGTGATGCTGCCAAGAACCAA GCTGCTTTGAACCCAAGAAACACTGTCTTCGATGCTAAGCGTTTGATTGGTAGAAGATTC GACGACGAATCTGTTCAAAAGGACATGAAGACCTGGCCTTTCAAGGTTATCGACGTCGAT GGTAACCCAGTCATCGAAGTCCAATACTTGGAAGAAACCAAGACTTTCTCCCCACAΆGAΆ ATTTCCGCTATGGTTTTGACCAAGATGAAGGAAATTGCTGAAGCTAAGATTGGTAAGAAG GTTGAAAAGGCCGTCATTACTGTCCCAGCTTACTTTAACGACGCTCAAΆGACAAGCTACC AAGGATGCCGGTGCCATTTCTGGTTTGAACGTTTTGCGTATCATCAACGAACCTACTGCC GCTGCTATTGCTTACGGTCTAGGTGCTGGTAAGTCCGAAAAGGAAAGACATGTTTTGATT TTCGATTTGGGTGGTGGTACTTTCGATGTTTCCTTGTTGCACATTGCTGGTGGTGTTTAC ACTGTTAAATCTACTTCCGGTAACACTCACTTGGGTGGTCAAGATTTCGACACCAACTTG TTGGAACACTTCAAGGCTGAATTCAAGAAGAΆGACTGGTTTGGACATCTCCGACGATGCC AGAGCTTTGAGAAGATTGAGAACTGCTGCTGAAAGAGCTAAGAGAACCTTATCTTCTGTC ACTCAAACTACCGTTGAAGTTGACTCTTTGTTTGACGGTGAAGATTTCGAATCCTCTTTG ACTAGAGCTAGATTTGAAGACTTGAACGCCGCATTGTTCAAGTCTACTTTGGAACCTGTT GAACAAGTTTTGAAGGATGCTAAGATCTCTAAGTCTCAAATCGACGAAGTTGTCTTGGTT GGTGGTTCCACCAGAATTCCAAAGGTCCAAAAGTTGTTGTCTGACTTCTTTGACGGTAAG CAATTGGAAAAATCTATTAACCCAGATGAAGCTGTTGCTTACGGTGCTGCTGTTCAAGGT GCTATCTTGACCGGCCAATCCACATCTGACGAAACCAAGGACTTGTTGTTGTTAGATGTT GCTCCATTATCTCTAGGTGTTGGTATGCAAGGTGACATGTTCGGTATCGTTGTTCCAAGA AACACTACTGTTCCAACCATCAAGAGAAGAACCTTTACTACATGTGCTGACAACCAAACC ACCGTTCAATTCCCAGTCTACCAAGGTGAACGTGTTAACTGTAAAGAAAΆCACTTTGTTG GGTGAΆTTCGACTTGAAGAACATCCCAATGATGCCAGCTGGTGAACCAGTCTTGGAAGCT ATCTTCGAAGTTGATGCTAACGGTATCTTGAAGGTTACTGCCGTCGAAAAGTCTACCGGT AAGTCTTCTAACATCACTATCTCTAACGCTGTTGGTAGATTGTCTTCTGAAGAAATTGAA AAGATGGTTAACCAAGCTGAAGAGTTCAAGGCTGCCGATGAAGCTTTTGCCAAGAAGCAC GAAGCTAGACAAAGATTGGAATCCTACGTTGCCTCCATCGAACAAACTGTCACTGACCCA GTCTTGTCTTCTAAATTGAΆGAGAGGTTCCAAGTCCAAGATTGAAGCTGCTTTGTCCGAT GCTTTGGCTGCTTTGCAAATCGAAGACCCATCTGCTGATGAATTGAGAAAGGCTGAAGTT GGTTTGAAGAGAGTTGTCACCAAGGCCATGTCTTCTCGTTAA
Further information on SSBl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000002388. It will be appreciated that, by "SSBl", we include fragments or variants thereof having equivalent S SBl -like activity.
SSB2 is another S. cerevisiae helper protein of interest for the present invention. Ssb2p is a cytoplasmic ATPase that is a ribosome-associated molecular chaperone. It may be involved in the folding of newly-synthesized polypeptide chains. It is a member of the heat shock protein 70 (HSP70) family and is a homolog of SSBl. A published protein sequence for the protein Ssb2p is as follows:
MAEGVFQGAIGIDLGTTYSCVATYESSVEIIANEQGNRVTPSFVAFTPQERLIGDAAKNQ AALNPRNTVFDAKRLIGRRFDDESVQKDMKTWPFKVIDVDGNPVIEVQYLEETKTFSPQE ISAMVLTKMKEIAEAKIGKKVEKAVITVPAYFNDAQRQATKDAGAISGLNVLRIINEPTA AAIAYGLGAGKSEKERHVLIFDLGGGTFDVSLLHIAGGVYTVKSTSGNTHLGGQDFDTNL LEHFKAEFKKKTGLDISDDARALRRLRTAAERAKRTLSSVTQTTVEVDSLFDGEDFESSL TRΆRFEDLNAALFKSTLEPVEQVLKDAKISKSQIDEWLVGGSTRIPKVQKLLSDFFDGK QLEKSINPDEAVAYGAAVQGAILTGQSTSDETKDLLLLDVAPLSLGVGMQGDIFGIWPR NTTVPTIKRRTFTTVSDNQTTVQFPVYQGERVNCKENTLLGEFDLKNIPMMPAGEPVLEA IFEVDANGILKVTAVEKSTGKSSNITISNAVGRLSSEEIEKMVNQAEEFKAADEAFAKKH EARQRLESYVASIEQTVTDPVLSSKLKRGSKSKIEAALSDALAALQIEDPSADELRKAEV GLKRWTKAMSSR*
SSB2 is encoded by anon-essential gene comprising an ORF that is 1.842 kbp in size and is located on chromosome XTV. Apublishednucleotide coding sequence of SSB2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
ATGGCTGAAGGTGTTTTCCAAGGTGCTATCGGTATCGATTTAGGTACAACATACTCTTGT GTTGCTACTTATGAATCTTCCGTTGAAATTATTGCCAACGAACAAGGTAACAGAGTTACT
CCATCTTTCGTTGCCTTCACCCCACAGGAAAGATTGATCGGTGATGCTGCCAΆGAACCAA
GCTGCTTTGAACCCAAGAAACACTGTTTTTGATGCTAAGCGTTTGATTGGTAGAAGATTC
GACGACGAGTCTGTCCAAAAGGACATGAAGACCTGGCCTTTCAAGGTTATCGACGTCGAT
GGTAACCCAGTCATTGAΆGTCCAATACTTGGAAGAAACCAAGACTTTCTCCCCACAAGAA ATTTCCGCTATGGTCTTGACCAAGATGAAGGAAATTGCTGAAGCTAAGATTGGTAAGAAG
GTTGAAAAGGCTGTCATTACTGTCCCAGCTTACTTTAACGΆTGCCCAAAGACAΆGCTACC AAGGATGCCGGTGCCATTTCTGGTTTGAACGTTTTGCGTATCATCAACGAACCTACTGCC GCTGCTATTGCTTACGGTCTAGGTGCTGGTAAGTCCGAAAAGGAAAGACATGTTTTGATT
TTCGATTTGGGTGGTGGTACTTTCGATGTTTCCTTGTTGCACATTGCTGGTGGTGTTTAC ACTGTTAAATCTACTTCCGGTAACACTCACTTGGGTGGTCAAGATTTCGACACCAACTTG TTGGAACACTTCAAGGCTGAATTCAAGAAGAAGACTGGTTTGGACATCTCCGACGATGCC AGAGCTTTGAGAAGATTGAGAACTGCTGCTGAAAGAGCTAAGAGAACCTTATCTTCTGTC ACTCAΆACTACCGTTGAΆGTTGACTCTTTGTTTGACGGTGAAGATTTCGAATCCTCTTTG ACTAGAGCTAGATTTGAΆGACTTGAACGCCGCATTGTTCAAGTCTACTTTGGAACCTGTT GAACAAGTTTTGAAGGATGCTAAGATCTCTAAGTCTCAAATCGACGAAGTTGTCTTGGTT GGTGGTTCTACCAGAATTCCAAAGGTCCAAAAGTTGTTGTCTGACTTCTTTGACGGTAAG CAATTGGAAAAATCTATTAACCCAGATGAAGCTGTTGCTTACGGTGCTGCTGTTCAΆGGT GCTATCTTGACTGGCCAATCCACATCTGACGAAACCAAGGACTTGTTGTTGTTAGATGTT GCTCCATTATCTCTAGGTGTTGGTATGCΆAGGTGACATTTTCGGTATTGTTGTCCCAAGA AACACAACTGTTCCAACCATCAAGAGAAGAACCTTCACAACTGTCAGTGACAΆCCΆAACC ACCGTTCAATTCCCAGTCTACCAAGGTGAACGTGTCAACTGTAAAGAAAACACTTTGTTG GGTGAATTCGACTTGAAGAACATCCCAATGATGCCAGCTGGTGAACCAGTCTTGGAAGCT ATCTTCGAAGTTGATGCTAACGGTATCTTGAAGGTTACTGCCGTCGAAAAGTCTACCGGT AAGTCTTCTΆΆCATCACTATCTCCAACGCTGTCGGTAGATTGTCTTCTGΆAGAAATTGAΆ AAGATGGTTAΆCCAAGCCGAAGAGTTCAAGGCTGCTGATGAAGCTTTTGCTAΆGAAGCAC GAAGCTAGACAAAGACTAGAATCCTACGTCGCTTCCATCGAACAAACCGTCACTGACCCA GTCTTGTCTTCTAAATTGAAGAGAGGTTCCAAGTCCAAGATCGAAGCTGCTTTGTCCGAT GCTTTGGCTGCTTTGCAAATCGAAGACCCATCCGCTGATGAGTTGAGAAΆGGCAGAAGTT GGTTTGAAGAGAGTTGTCACCAAGGCCATGTCTTCTCGTTAA
Further information on SSB2 can be obtained from the URL address hftp://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000005153.
It will be appreciated that, by "SSB2", we include fragments or variants thereof having equivalent SSB2-like activity.
ECMlO is another S. cerevisiae helper protein of interest for the present invention and is also known as SSC3. EcmlOp is a heat shock protein of the Hsp70 family, which is localised in mitochondrial nucleoids. It is thought to play a role in protein translocation. It interacts with Mgelp in an ATP-dependent manner. Over-expression has been shown to induce extensive mitochondrial DNA aggregations. A published protein sequence for the protein EcmlOp is as follows: MLPSWKAFKAHNILRILTRFQSTKIPDAVIGIDLGTTNSAVAIMEGKVPRIIENAEGSRT TPSVVAFTKDGERLVGEPAKRQSVINSENTLFATKRLIGRRFEDAEVQRDINQVPFKIVK HSNGDAWVEARNRTYSPAQIGGFILNKMKETAEAYLAKSVKNAVVTVPAYFNDAQRQATK DAGQIIGLNVLRVVNEPTAAALAYGLDKSEPKVIAVFDLGGGTFDISILDIDNGIFEVKS TNGDTHLGGEDFDIYLLQEIISHFKKETGIDLSNDRMAVQRIREAAEKAKIELSSTLSTE INLPFITADAAGPKHIRMPFSRVQLENITAPLIDRTVDPVKKALKDARITASDISDVLLV GGMSRMPKVADTVKKLFGKDASKAVNPDEAVALGAAIQAΆVLSGEVTDVLLLDVTPLSLG IETLGGVFTKLIPRNSTIPNKKSQIFSTAASGQTSVEVKVFQGERELVKDNKLIGNFTLA GIPPAPKGTPQIEVTFDIDANGIINVSAKDLASHKDSSITVAGASGLSDTEIDRMVNEAE RYKNQDRARRNAIETANKADQLANDTENSIKEFEGKLDKTDSQRLKDQISSLRELVSRSQ AGDEVNDDDVGTKIDNLRTSSMKLFEQLYKNSDNPETKNGRENK*
ECMlO is encoded by a non-essential gene comprising an ORF that is 1.935 kbp in size and is located on chromosomeV. Apublishednucleotide coding sequence ofECMlO is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGTTACCATCATGGAAAGCCTTTAAAGCACATAATATACTTCGTATTCTGACCCGTTTC CAGTCAACCAAAATTCCAGATGCAGTTATCGGTATTGATTTAGGTACTACCAATTCTGCG GTAGCTATTATGGAAGGTAAAGTTCCGAGAATTATCGAAΆATGCAGAAGGCTCAΆGAACT ACTCCGTCTGTAGTGGCTTTCACTAΆAGACGGAGAΆCGTTTAGTTGGTGAGCCAGCCAAΆ CGACAATCCGTCATAAACTCAGAAAACACTTTGTTTGCTACTAAGCGTTTAATCGGCCGC CGTTTCGAGGACGCTGAΆGTCCAAAGAGATATTAATCAGGTTCCTTTCAAAATCGTCAAG CATTCTAATGGAGATGCCTGGGTAGAGGCTAGAAACAGAACGTACTCCCCCGCCCAAATA GGAGGTTTTATCTTAAATAΆAΆTGAAGGAAACAGCGGAGGCTTACTTAGCGAAGAGCGTC AAAAATGCTGTTGTCACCGTTCCTGCTTACTTCAATGATGCCCAAAGACAAGCTACTAAA GACGCAGGACAAATTATTGGGCTTAATGTATTACGTGTTGTCAACGAACCAΆCAGCTGCT GCCCTAGCTTACGGTCTAGATAAATCAGAGCCAAAAGTCATTGCTGTTTTCGACTTGGGC GGTGGTACTTTCGATATTTCAATCCTGGACATCGATAACGGTATCTTTGAGGTTAAATCT ACCAATGGTGACACCCATTTGGGTGGCGAAGATTTTGACATTTATTTGTTGCAAGAAATT ATTTCTCATTTCAAGAAAGAAACCGGTATCGATTTGAGTAATGACCGTATGGCTGTCCAA AGAATAΆGAGAAGCCGCTGAAΆAGGCTAAAATCGAACTGTCTTCTACACTCTCTACAGAA ATAAACTTGCCTTTCATAACTGCTGATGCTGCAGGCCCAAAGCATATTCGTATGCCCTTT TCTAGGGTTCAGCTTGAGAATATAACCGCCCCATTGATTGATAGAACGGTTGATCCTGTC AAΆAAAGCACTGAAAGACGCAAGAATTACCGCCTCAGATATATCGGATGTTTTATTAGTT GGTGGTATGTCAAGGATGCCCAAGGTTGCAGATACTGTAAAGAAATTATTCGGTAAGGAT GCATCAAΆAGCTGTTAACCCTGATGAAGCAGTCGCTTTAGGGGCCGCTATACAGGCTGCG GTCTTGTCTGGTGAAGTTACCGATGTTTTGTTGCTAGATGTCACTCCCCTATCATTGGGT
ATTGAAACTTTAGGAGGAGTTTTTACAAAATTAATCCCAAGAAATTCTACAATTCCCAAT AAGAAATCTCAAATTTTTTCAACTGCGGCATCAGGTCAAACATCGGTGGAAGTTAAAGTT TTCCAAGGTGAGAGGGAGTTAGTCAAGGATAACAAATTAATAGGTAATTTTACTCTTGCG GGCATTCCTCCAGCTCCAAAAGGTACCCCACAAATTGAAGTCACTTTTGATATCGATGCG AACGGCATCATCAACGTTTCAGCAΆΆAGATCTCGCCAGCCACAAAGACTCTTCCATCACT GTTGCCGGAGCGTCTGGGCTATCTGATACGGAGATTGATCGAATGGTTAATGAAGCGGAA AGATATAAΆAATCAGGATAGAGCCAGAAGGAATGCCATCGAAACCGCTAACAAAGCTGAC CAGCTAGCTAATGACACAGAΆAATTCCATTAAGGAATTCGAAGGTAΆGCTAGATAAΆACT GATTCTCAAAGACTAAAAGATCAAATTTCATCCTTAAGGGAATTGGTTTCTCGGAGTCAA GCTGGAGATGAGGTTAATGATGACGATGTTGGAACAAAAATTGACAATTTGCGAACTTCA TCGATGAAACTTTTTGAΆCAGTTATACAΆGAACAGTGACAATCCTGAAACTAAGAACGGG AGAGAAAATAAATAA
Further information on ECMlO can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000756.
It will be appreciated that, by "ECMlO", we include fragments or variants thereof havingequivalentECMlO-like activity.
MDJl is another S. cerevisiae helper protein of interest for the present invention. Mdj Ip is a protein involved in folding of mitochondrially synthesised proteins in the mitochondrial matrix. It localises to the mitochondrial inner membrane and is a member of the DnaJ family of molecular chaperones. A published protein sequence for the protein Mdj Ip is as follows:
MAFQQGVLSRCSGVFRHHVGHSRHINNILYRHAIAFASIAPRIPKSSFHTSAIRNNEAFK DPYDTLGLKKSATGAEIKKAYYKLAKKYHPDINKEPDAE KKFHDLQNAYEILSDETKRQQ YDQFGPAAFGGGGAAGGAGGGSGSPFGSQFHDFSGFTSAGGSPFGGINFEDLFGAAFGGG GRGSGGASRSSSMFRQYRGDPIEIVHKVSFKDAVFGSKNVQLRFSALDPCSTCSGTGMKP NTHKVSCSTCHGTGTTVHIRGGFQMMSTCPTCNGEGTMKRPQDNCTKCHGEGVQVNRAKT ITVDLPHGLQDGDWRIPGQGSYPDIAVEADLKDSVKLSRGDILVRIRVDKDPNFSIKNK YDIWYDKEIPITTAALGGTVTIPTVEGQKIRIKVAPGTQYNQVISIPNMGVPKTSTIRGD MKVQYKIWKKPQSLAEKCLWEALADVTNDDMAKKTMQPGTAAGTAINEEILKKQKQEEE KHAKKDDDNTLKRLENFITNTFRKIKGDKKN* MDJl is encoded by a non-essential gene comprising an ORF that is 1.536 kbp in size and is located on chromosome VI. A published nucleotide coding sequence ofMDJl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGGCTTTCCAACAAGGTGTATTGTCAAGGTGTTCCGGTGTCTTTAGACACCATGTGGGA CATTCTCGCCATATCAATAATATTCTTTATAGACATGCCATCGCGTTTGCATCCATGGCT CCACGAATACCAAAΆTCTAGCTTCCATACTTCTGCAΆTCAGAAACAΆCGAAGCATTCAAG GACCCGTACGATACTTTAGGCTTGAAGAAATCTGCTACAGGTGCGGAAATCAAAAAAGCA TACTACAAACTGGCAAAGAAGTACCACCCGGATATCAACAAGGAACCGGATGCTGAGAAG AΆATTCCACGATTTACAGAACGCTTATGAAATTCTGTCAGACGAAACGAAGAGGCAGCAG TACGATCAATTTGGGCCCGCTGCCTTCGGCGGCGGCGGTGCCGCTGGAGGTGCCGGTGGT GGTAGTGGCTCTCCCTTTGGTTCCCAATTTCATGATTTCTCAGGATTCACCAGTGCAGGC GGCTCGCCATTTGGCGGTATCAATTTTGAAGACCTGTTTGGTGCTGCATTTGGTGGTGGT GGCCGCGGTAGCGGTGGCGCAAGCAGGTCGTCATCTATGTTCAGACAATATAGGGGCGAC CCAATCGAGATTGTCCATAAAGTGTCTTTCAAGGACGCAGTGTTTGGGTCCAAGAΆCGTT CAGTTAAGATTCTCTGCGCTGGACCCTTGTAGTACCTGTTCAGGGACGGGAATGAΆΆCCA AΆCACGCATAAGGTCAGTTGTAGCACTTGTCACGGAACAGGAACCACTGTTCACATTAGG GGCGGATTTCAGATGATGTCGACTTGTCCTACTTGCAACGGTGAΆGGTACCATGAΆΆCGG CCTCAGGACAATTGTACCAΆGTGCCATGGTGAGGGTGTTCAGGTCAACAGGGCAAAGACA ATTACGGTGGACTTGCCACATGGATTACAGGACGGCGACGTGGTCAGGATCCCTGGCCAA GGCTCATACCCTGACATCGCTGTAGAGGCGGACTTGAAAGATTCAGTCAAGTTATCAΆGA GGTGATATTTTGGTGAGAΆTTCGTGTCGACAAGGATCCCAACTTTTCGATAΆAGAACAAG TACGATATTTGGTACGACAAGGAGATTCCTATAACCACAGCTGCACTTGGTGGTACTGTC ACTATCCCCACTGTGGAGGGACAAAAGATCAGGATAAAGGTCGCTCCAGGGACTCAATAC AATCAΆGTGATATCCATTCCTAACATGGGTGTTCCTAAΆACATCAΆCCATTCGCGGTGAT
ATGAAAGTCCAGTACASLGATCGTTGTTAAGAAACCGCAATCGCTGGCAGAAAAATGCTTG
TGGGAGGCACTGGCAGATGTCACCAΆCGATGACATGGCCAAGΆAAACCATGCAΆCCGGGC ACAGCCGCGGGTACAGCCATTAATGAΆGAGATACTGAΆGAAACAAAAΆCAAGAAGAGGAA ■ AΆACACGCAΆAAAAGGATGACGACAΆCACTTTGAAGAGACTAGAΆAATTTCATTACCAΆC ACATTCAGGAAGATCAAAGGTGACAAAAAAAATTAA
Further information on MDJl can be obtained from - the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=SOOOOO 1878. It will be appreciated that, by "MDJl", we include fragments or variants thereof having equivalent MDJl -like activity.
MD J2 is another S. cerevisiae helper protein of interest for the present invention. Mdj2p is a protein of the mitochondrial inner membrane. Its function partially overlaps that of Mdjlp, which is a chaperone involved in folding of mitochondrially synthesised proteins in the mitochondrial matrix. It is a member of the DnaJ family. A published protein sequence for the protein Mdj2p is as follows:
MVLPIIIGLGVTMVALSVKSGLNAWTVYKTLSPLTIAKLNNIRIENPTAGYRDALKFKSS LIDEELKNRLNQYQGGFAPRMTEPEALLILDISAREINHLDEKLLKKKHRKAMVRNHPDR GGSPYMAAKINEAKEVLERSVLLRKR*
MDJ2 is encoded by a non-essential gene comprising an ORF that is 0.441 kbp in size and is located on chromosome XTV. A published nucleotide coding sequence of MDJ2 is as follows, 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:
ATGGTTTTGCCTATAATAATTGGTTTGGGCGTGACAATGGTTGCTCTAAGTGTCAAGTCT GGTCTCAATGCATGGACCGTCTACAAGACCCTGTCCCCTTTAACTATTGCAAAACTAAAT AACATTCGCATAGAAAACCCGACGGCGGGCTACCGCGATGCACTTAAGTTCAAAΆGCTCA CTGATAGACGAAGAACTGAAAAATAGATTAAACCAGTACCAGGGAGGCTTTGCACCGCGA ATGACAGAGCCCGAAGCCTTGCTCATCTTGGATATCTCCGCCAGAGAGATTAATCACTTG GATGAAAAATTACTGAAAAAAAAGCACAGGAAGGCTATGGTTCGTAACCACCCAGACAGA GGAGGGAGTCCCTACATGGCGGCCAAGATAAATGAGGCGAAAGAΆGTTCTCGAAAGAAGT GTTTTACTAAGAAAGAGATAA
Further information on MDJ2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000005272.
It will be appreciated that, by "MDJ2", we include fragments or variants thereof having equivalentMDJ2-like activity. EROl is another S. cerevisiae helper protein of interest for the present invention. Erolp is a glycoprotein required for oxidative protein folding in the endoplasmic reticulum. A published protein sequence for the protein Erolp is as follows:
MRLRTAIATLCLTAFTSATSNNSYIATDQTQNAFNDTHFCKVDRNDHVSPSCNVTFNELN AINENIRDDLSALLKSDFFKYFRLDLYKQCSFWDANDGLCLNRACSVDVVEDWDTLPEYW QPEILGSFNNDTMKEADDSDDECKFLDQLCQTSKKPVDIEDTINYCDVNDFNGKNAVLID LTANPERFTGYGGKQAGQIWSTIYQDNCFTIGETGESLAKDAFYRLVSGFHASIGTHLSK EYLNTKTGKWEPNLDLFMARIGNFPDRVTNMYFNYAVVAKALWKIQPYLPEFSFCDLVNK EIKNKMDNVISQLDTKIFNEDLVFANDLSLTLKDEFRSRFKNVTKIMDCVQCDRCRLWGK IQTTGYATALKILFEINDADEFTKQHIVGKLTKYELIALLQTFGRLSESIESVNMFEKMY GKRLNGSENRLSSFFQNNFFNILKEAGKSIRYTIENINSTKEGKKKTNNSQSHVFDDLKM PKAEIVPRPSNGTVNKWKKAWNTEVNNVLEAFRFIYRSYLDLPRNIWELSLMKVYKFWNK FIGVADYVSEETREPISYKLDIQ*
EROl is encodedby an essential gene comprising an ORP thatis 1.692 kbp in size and is located on chromosome XIII. A published nucleotide coding sequence of EROl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGAGATTAAGAACCGCCATTGCCACACTGTGCCTCACGGCTTTTACATCTGCAACTTCA AACAATAGCTACATCGCCACCGACCAAACACAAAATGCCTTTAATGACACTCACTTTTGT AAGGTCGACAGGAATGATCACGTTAGTCCCAGTTGTAACGTAACATTCAATGAATTAAAT GCCATAAΆTGAAAACATTAGAGATGATCTTTCGGCGTTATTAAAΆTCTGATTTCTTCAAΆ TACTTTCGGCTGGATTTATACAAGCAATGTTCATTTTGGGACGCCAACGATGGTCTGTGC TTAAACCGCGCTTGCTCTGTTGATGTCGTAGAGGACTGGGATACACTGCCTGAGTACTGG CAGCCTGAGATCTTGGGTAGTTTCAΆTAATGATACAΆTGAAGGAAGCGGATGATAGCGAT GACGAATGTAAGTTCTTAGATCAACTATGTCAAACCAGTAΆΆAAACCTGTAGATATCGAΆ GACACCATCAACTACTGTGATGTAAATGACTTTAACGGTAAAAACGCCGTTCTGATTGAT TTAACAGCAAATCCGGAACGATTTACAGGTTATGGTGGTAAGCAΆGCTGGTCAAATTTGG TCTACTATCTACCAAGACAACTGTTTTACAATTGGCGAAACTGGTGAATCATTGGCCAAA GATGCATTTTATAGACTTGTATCCGGTTTCCATGCCTCTATCGGTACTCACTTATCAAAG GAATATTTGAACACGAAΆACTGGTAAATGGGAGCCCAATCTGGATTTGTTTATGGCAΆGA ATCGGGAACTTTCCTGATAGAGTGACAAACATGTATTTCAATTATGCTGTTGTAGCTAAG GCTCTCTGGAAAATTCAACCATATTTACCAGAATTTTCATTCTGTGATCTAGTCAATAAA GAAATCAAAAΆCAAAATGGATAACGTTATTTCCCAGCTGGACACAAAAATTTTTAACGAΆ GACTTAGTTTTTGCCAACGACCTAΆGTTTGACTTTGAΆGGACGAATTCAGATCTCGCTTC AAGAATGTCACGAAGATTATGGATTGTGTGCAATGTGATAGATGTAGΆTTGTGGGGCAAΆ ATTCAAACTACCGGTTACGCAACTGCCTTGAAAATTTTGTTTGAAATCAACGACGCTGAT GAATTCACCAAACAACATATTGTTGGTAAGTTAACCAAΆTATGAGTTGATTGCACTATTA CAGACTTTCGGTAGATTATCTGAATCTATTGAATCTGTTAACATGTTCGAAAAAATGTAC GGGAAAAGGTTAAACGGTTCTGAAΆACAGGTTAΆGCTCATTCTTCCAAAATAΆCTTCTTC AACATTTTGAAGGAGGCAGGCAAATCGATTCGTTACACCATAGAGAACATCAATTCCACT AAΆGAAGGAAAGAAAAAGACTAACAATTCTCAATCACATGTATTTGATGATTTAAAAATG CCCAAAGCAGAAATAGTTCCAAGGCCCTCTAACGGTACAGTAAATAAATGGAAGAAΆGCT TGGAATACTGAAGTTAACAACGTTTTAGAAGCATTCAGATTTATTTATAGAAGCTATTTG GATTTACCCAGGAΆCATCTGGGAATTATCTTTGATGAAGGTATACAΆΆTTTTGGAATAΆA TTCATCGGTGTTGCTGATTACGTTAGTGAGGAGACACGAGAGCCTATTTCCTATAAGCTA GATATACAATAA
Further information on EROl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000004599.
It will be appreciated that, by "EROl", we include fragments or variants thereof having equivalent EROl-like activity.
ERV2 is another S. cerevisiae helper protein ofinterest for the present invention.
Erv2p is a flavin-linked sulfhydryl oxidase localisedto the endoplasmic reticulum lumen, involved in disulphide bond formationwithinthe ER. A published protein sequence forthe proteinErv2p is as follows:
MKQIVKRSHAIRIVAALGIIGLWMFFSSNELSIATPGLIKAKSGIDEVQGAAAEKNDARL
KEIEKQTIMPLMGDDKVKKEVGRASWKYFHTLLARFPDEPTPEEREKLHTFIGLYAELYP
CGECSYHFVKLIEKYPVQTSSRTAAΆMWGCHIHNKVNEYLKKDIYDCATILEDYDCGCSD
SDGKRVSLEKEAKQHG*
ERV2 is encoded by a non-essential gene comprising an ORF that is 0.591 kbp in size, located on chromosome XVI. A published nucleotide coding sequence of ERV2 is as follows, 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: ATGAAACAGATAGTCAAAAGAAGCCATGCCATCAGAATAGTTGCAGCATTAGGAATCATA
GGCCTGTGGATGTTTTTCTCGTCTAATGAACTATCCATCGCTACGCCGGGCCTAΆTCAAG
GCGAAGTCTGGTATAGATGAAGTGCAAGGGGCGGCTGCTGAGAAGAACGACGCTCGGTTG
AAAGAGATCGAGAAGCAAΆCCATTATGCCATTGATGGGCGATGACAAGGTGΆAGAAGGAA GTGGGCAGGGCGTCGTGGAAGTACTTCCATACCCTGCTGGCCCGTTTTCCGGACGAGCCT
ACTCCTGAAGAAAGAGAGAAACTGCACACGTTTATTGGGTTGTATGCAGAACTCTATCCA
TGCGGGGAATGTTCATATCACTTTGTAAAGTTGATTGAGAAGTATCCCGTACAGACATCT
AGCAGGACGGCTGCCGCAATGTGGGGΆTGCCACATTCACAACAAGGTGAACGAATACCTA
AAGAAAGACATATATGACTGTGCTACCATCCTGGAGGACTACGATTGTGGATGTAGTGAC AGCGACGGTAAACGCGTGTCTCTCGAGAAGGAGGCTAAACAGCACGGTTGA
Further information on ERV2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000006241.
It will be appreciated that, by "ERV2", we include fragments or variants thereof having equivalent ERV2-like activity.
EUGl is another S. cerevisiae helper protein of interest for the present invention. Euglp is a protein disulphide isomerase of the endoplasmic reticulum lumen, with an overlapping function with Pdilp. It may interact with nascent polypeptides in the ER. A published protein sequence for the protein Euglp is as follows:
MQVTTRFISAIVSFCLFASFTLAENSARATPGSDLLVLTEKKFKSFIESHPLVLVEFFAP WCLHSQILRPHLEEAASILKEHNVPWQIDCEANSMVCLQQTINTYPTLKI FKNGRI FDG QVYRGVKITDEITQYMI QLYEASVIYLNSEDEIQPYLENATLPWINRGLTGLNETYQEV ALDLAEDYVFLSLLDSEDKSLSIHLPNTTEPILFDGNVDSLVGNSVALTQWLKWILPYF TDIEPDLFPKYISSNLPLAYFFYTSEEELEDYTDLFTQLGKENRGQINFIALNSTMFPHH VRFLNMREQFPLFAIHNMINNLKYGLPQLPEEEYAKLEKPQPLDRDMIVQLVKDYREGTA KPIVKSEEIPKEQKSNVYKIVGKTHDDIVHDDDKDVL VKYYATWCIHSKRFAPIYEEIAN VLASDESVRDKILIAEVDSGANDILSFPVTGYPTIALYPAGNNSKPIIFNKIRNLEDVFE FIKESGTHHIDGQAIYDKLHQAKDSEVSTEDTVHDEL*
EUGl is encoded by a non-essential gene comprising an ORF that is 1.554 kbp in size and is located on chromosome IV. A published nucleotide coding sequence of EUGl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidentical proteinproduct:
ATGCAAGTGACCACAAGATTTATATCTGCGATAGTCTCGTTTTGCCTGTTTGCTTCTTTC ACGTTGGCTGAAAACAGCGCAAGAGCTACGCCGGGATCAGATTTACTCGTTCTAACAGAG AAGAΆATTTAΆΆTCATTCATCGAΆTCTCATCCGTTAGTCCTCGTCGAGTTTTTTGCTCCA TGGTGTTTGCATTCTCAGATCTTACGCCCTCACTTAGAAGAGGCCGCCTCTATTTTAAΆG GAGCATAACGTCCCAGTTGTTCAAATTGATTGTGAGGCTAACAGTATGGTTTGCCTGCAA CAAACTATAAΆTACCTACCCAACCTTGAAAATCTTTAAAAATGGTCGTATTTTTGATGGT CAAGTCTATCGCGGTGTCAAGATCACCGATGAAATCACTCAGTACATGATTCAGCTATAC GAGGCTTCTGTCATTTATTTAAATTCCGAΆGATGAAATCCAACCATACTTGGAΆAATGCA ACTTTACCAGTAGTAATAAΆCAGAGGCTTGACAGGCTTGAATGAAACGTATCAAGΆAGTC GCACTGGACCTTGCTGAGGATTACGTCTTTTTATCCCTTCTAGATTCAGAΆGATAAGTCA TTATCAATCCACTTGCCAAACACTACAGAACCAATTCTGTTTGATGGAAATGTAGACTCT TTGGTCGGAAATTCCGTTGCTCTAACTCAGTGGTTAAAAGTGGTAATTTTACCTTACTTT ACCGACATCGAACCTGATCTCTTCCCCAAGTACATTTCTAGCAATTTGCCGTTGGCTTAC TTCTTTTATACTTCTGAGGAAGAATTGGAAGATTACACTGATCTTTTCACGCAGTTAGGT AAGGAAAATCGTGGCCAAATAAATTTCATTGCATTAAACTCTACAATGTTCCCACACCAC GTTAGATTCCTAΆATATGAGAGAACAGTTCCCATTATTTGCTATCCATAATATGATCAAT AATCTGAAΆTATGGTTTACCACAACTACCAGAAGAΆGAGTACGCGAAATTAGAAAAACCA CAACCACTAGACAGAGATATGATCGTTCAGTTGGTAAAAGATTACCGTGAAGGTACTGCC AAGCCAΆTTGTTAAGTCAGAAGAGATTCCAAAAGAΆCAAAAGTCCAATGTTTATAAAATA GTTGGGAAGACACATGACGACATTGTTCATGATGATGACAAGGATGTCCTTGTCAAATAT TACGCGACATGGTGTATTCATAGTAAAAGGTTTGCGCCTATTTACGAAGAAATTGCAAAT GTCTTAGCATCTGATGAATCTGTTCGCGATAAAATCTTGATCGCCGAAGTAGATTCAGGG GCAAATGATATCTTAAGTTTTCCTGTGACΆGGATATCCAACCATTGCTTTGTATCCTGCC GGAAATAACTCTAAGCCTATTATCTTCAATAAAATTAGAAATTTGGAAGATGTTTTCGAA
TTTATCAAGGAATCAGGTACACATCACATTGACGGCCAGGCAATTTATGATAAATTGCAC CAGGCCAAGGATTCTGAAGTGTCTACTGAAGATACCGTACATGATGAATTATAA
Further information on EUGl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/srnglepageformat?sgdid=S000002926.
It will be appreciated that, by "EUGl", we include fragments or variants thereof having equivalent EUGl -like activity. MPDl is another S. cerevisiae helper protein ofinterest for the present invention. Mpdlp is a member ofthe protein disulphide isomerase (PDI) family. Its over- expression suppresses the defectinmaturation ofcarboxypeptidase Y, and defects in other essential Pdilp functions that can be caused by PDIl deletion. A publishedprotein sequence for theproteinMpdlp is as follows:
MLFLNIIKLLLGLFIMNEVKAQNFYDSDPHISELTPKSFDKAIHNTNYTSLVEFYAPWCG HCKKLSSTFRKAAKRLDGVVQVAAVNCDLNKNKALCAKYDVNGFPTLMVFRPPKIDLSKP IDNAKKSFSAHANEVYSGARTLAPIVDFSLSRIRSYVKKFVRIDTLGSLLRKSPKLSWL FSKQDKISPVYKSIALDWLGKFDFYSISNKKLKQLTDMNPTYEKTPEIFKYLQKVIPEQR QSDKSKLVVFDADKDKFWEYEGNSINKNDISKFLRDTFSITPNEGPFSRRSEYIAYLKTG KKPIKKNHSSSGNKHDEL*
MPDl is encoded by anon-essential gene comprising an ORF that is 0.957 kbp in size and is located on chromosome XV. A published nucleotide coding sequence ofMPDl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
ATGTTATTTCTTAATATTATTAAGCTCCTTTTGGGACTTTTTATTATGAATGAAGTAAAG GCGCAAAACTTTTACGATTCCGATCCTCATATATCAGAGTTAACGCCAAAAAGCTTCGAT AΆΆGCGATCCATAACACAAATTACACATCATTAGTGGAATTTTATGCTCCGTGGTGCGGC CATTGTAAGAAGCTCTCTAGTACGTTCCGCAAGGCAGCAAAΆAGATTGGATGGTGTAGTC CAAGTTGCTGCTGTAAΆCTGTGACCTTAACAΆGAATAAGGCTTTGTGTGCTAAΆTACGAC GTAAACGGATTTCCCACGTTAATGGTATTTAGGCCCCCAAAAATTGACCTATCTAAGCCA ATAGATAΆCGCCAAAAAAAGTTTCΆGCGCTCATGCCAATGAAGTGTACTCAGGTGCAAGA ACTCTCGCGCCTATTGTTGATTTTTCTCTTTCAΆGAATAAGGTCATATGTCAAΆAAGTTT GTCCGTATAGATACACTTGGCTCTTTACTTAGAAAGTCACCCAAACTTTCCGTGGTGTTG TTTTCCAAACAAGACAAAATTTCACCGGTTTATAAAAGCATTGCCCTTGATTGGTTAGGA AAGTTCGAT.TTTTATTCAATTTCAAACAAAAAACTCAAGCAACTAACCGATATGAACCCA ACATATGAAAAAACTCCTGAGATTTTCAAATATTTGCAGAAGGTCATTCCTGAACAGCGA CAGAGCGATAAAAGTAAGCTTGTCGTTTTTGATGCAGACAAAGATAAATTTTGGGAGTAT GAAGGGAΆCTCAATCAACAΆAAATGACATTTCCAAATTTCTGCGGGACACTTTTAGTATT ACCCCCAATGAGGGTCCTTTTAGTAGACGTTCTGAATATATTGCTTACTTAΆAAΆCTGGC AAGAAGCCAATTAAAAAGAACCATTCCTCCTCAGGAAACAAGCACGACGAATTGTAG Further information on MPDl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000005814.
It will be appreciated that, by "MPDl", we include fragments or variants thereof having equivalent MPD 1 -like activity.
MPD2 is another S. cerevisiae helper protein of interest for the present invention. Mpd2p is a member of the protein disulphide isomerase (PDI) family. It exhibits chaperone activity. Its overexpression suppresses the lethality of a PDIl deletion but does not complement all Pdilp functions. It undergoes oxidation by Erolp. A published protein sequence for the protein Mpd2p is as follows:
MKLHGFLFSVLSTCWIL'PALAYSEAVTMVKSIEQYFDICNRNDSYTMIKYYTSWCQHCK TLAPVYEELGELYAKKANKDDTPINFLEVNCEFFGPTLCTDLPGFPIIELVKPRTKPLVL PKLDWSSMKFHERLWQRIKTWFNNPKYQLDTSRVVRFEGSRNLKSLSNFIDTVRSKDTEE : RFIEHIFDDSRNCNEELRSQQLLCKAGKEYYSDTLSKLYGDVNGLEKE RRRLEALIKQNG DDLSKEVKEKLKIIRLQLSLLSHIEDQLEDTSSHDEL*
MPD2 is encoded by a non-essential gene comprising an ORF that is 0.834kbp in size and is located on chromosome XV. A published nucleotide coding sequence of MPD2 is as follows, 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:
ATGAAATTGCACGGCTTTTTATTTTCCGTATTATCAACATGCGTCGTCATTTTACCAGCG TTGGCCTACAGTGAAGCTGTCACGATGGTCAAGTCGATTGAGCAGTACTTCGATATCTGC AATAGGAATGATTCTTACACAATGATAΆAATACTACACTTCTTGGTGCCAACATTGTAAΆ ACTCTGGCCCCAGTATACGAAGAGCTTGGTGAGCTATACGCCAAAAAAGCTAATAAAGAT GATACCCCAATTAACTTCCTTGAΆGTTAACTGTGAATTCTTCGGGCCΆACTTTATGTACC GACTTGCCTGGATTTCCAATAATTGAACTGGTCAAACCTCGTACTAAGCCCTTAGTTCTT CCGAAGCTCGATTGGTCGTCTATGAAATTTCATGAAAGACTATGGCAΆAGAATCAΆGACG TGGTTCAACAATCCTAAGTACCAACTGGATACGTCTAGGGTTGTTCGTTTTGAAGGGAGT AGGAΆCCTAΆAGAGTTTAAGCAACTTTATCGATACTGTAAGAΆGTAAA'GATACAGAAGAA AGATTCATAGAACATATTTTCGATGATTCTAGGAATTGCAATGAAGAATTACGTTCTCAA CAGCTTCTGTGTAAAGCTGGTAAAGAATACTACTCTGATACTTTATCTAAATTATACGGT GACGTGAΆTGGGCTGGAAΆAGGAAAGGCGAAGACTAGAAGCTTTAATTAAGCAAAATGGA GATGACTTGAGTAAAGAAGTTAAΆGAΆΆAΆCTGAΆAATCATTCGTCTACAATTGAGCCTA TTATCACACATAGAAGACCAGTTAGAAGATACCAGTAGTCATGACGAGCTTTGA
Further information on MPD2 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000005448.
It will be appreciated that, by "MPD2", we include fragments or variants thereof having equivalent MPD2-like activity.
EPSl is another S. cerevisϊae helper protein of interest for the present invention. Eps Ip is a Pdilp (protein disulphide isomerase)-related protein involved in endoplasmic reticulum retention of resident ER proteins. A published protein sequence for the protein Eps Ip is as follows:
MKMNLKRL VVTFFSCITFLLKFTIAAAEPPEGFPEPLNPTNFKEELSKGLHIIDFYSPYC PHCKHLAPVWMETWEEFKEESKTLNITFSQVNCIESADLCGDENIEYFPEIRL YNPSGYI KSFTETPRTKESLIAFARRESMDPNNLDTDLDSAKSESQYLEGFDFLELIAGKATRPHLV SFWPTKDMKNSDDSLEFKMCDKCHEFQRTWKIISRQLAVDDINTGHVNCESNPTICEELG FGDLVKITNHRADREPKVALVLPNKTSNNLFDYPNGYSAKSDGYVDFARRTFTNSKFPNI TEGELEKKANRDIDFLQERGRVTNNDIHLVFSYDPETVVIEDFDILEYLIEPLSKIPNIY LHQIDKNLINLSRNLFGRMYEKINYDASQTQKVFNKEYFTMNTVTQLPTFFMFKDGDPIS YVFPGYSTTEMRNIDAIMDWVKKYSNPLVTEVDSSNLKKLISFQTKSYSDLAIQLISSTD HKHIKGSNKLIKNLLLASWEYEHIRMENNFEEINERRARKΆDGIKKIKEKKAPANKIVDK MREEIPHMDQKKLLLGYLDISKEKNFFRKYGITGEYKIGDVIIIDKSNNYYYNKDNFGNS LTSNNPQLLREAFVSLNIPSKALYSSKLKGRLINSPFHNVLSFLDIIHGNGMPGYLIVIV LFIAILKGPS I YRRYKVRKHYRAKRNAVGILGNMEKKKNQD*
EPSl is a non-essential gene comprising an ORF that is 2.106 kbp in size and is located on chromosome DC A published nucleotide coding sequence of EPSl is as follows, 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:
ATGAAAATGAATCTGAAAAGGCTCGTAGTTACCTTCTTCTCATGCATCACCTTTCTGCTG AAATTCACTATAGCCGCCGCTGAACCACCAGAGGGCTTTCCAGAGCCCTTAAATCCAACA
AACTTCAAAGAAGAGCTATCTAAGGGGCTGCATATTATTGACTTCTATAGTCCATACTGT CCGCACTGCAAACATTTAGCACCTGTTTGGATGGAAΆCATGGGAGGAGTTTAAAGAGGΆG AGCAAAACACTGAACATAACATTTTCACAGGTTAACTGCATCGAGAGCGCCGATTTGTGT GGAGATGAAΆATATTGAATACTTCCCTGAAATTAGACTTTATAACCCCTCAGGATACATC AAATCGTTCACTGAAACACCGAGGACCAAAGAATCATTAATTGCATTTGCACGCAGGGAG TCTATGGACCCAAATAACCTCGATACTGATCTGGATTCTGCTAAAAGTGAGAGCCAGTAT CTCGAAGGCTTTGATTTTCTCGAGCTGATCGCTGGTAAGGCGACTAGGCCACATTTGGTT TCCTTCTGGCCAACAAAAGATATGAAAAATAGCGATGATTCACTAGAATTCAAAAACTGT GACAAΆTGCCATGAATTCCAAΆGGACTTGGAAGATCATTTCAAGACAGTTAGCCGTGGAT GATATCAACACGGGCCACGTTAATTGCGAATCTAATCCAACAATCTGTGAAGAACTGGGC TTTGGCGACTTGGTGAAAATAACCAACCACAGAGCCGATAGAGAACCCAAGGTAGCATTA GTCCTACCCAATAAAΆCCTCAAATAATTTGTTCGACTATCCCAATGGCTACTCAGCGAAG TCAGATGGCTATGTAGATTTTGCCAGGAGGACTTTTACAAACAGTAAATTTCCCAATATA ACAGAAGGGGAGCTCGAAΆAAAAΆGCAAΆCAGAGACATTGATTTTCTGCAAGAAAGGGGA CGAGTAACTAATAATGATATCCATTTAGTTTTTTCATATGACCCCGAAACTGTTGTTATT GAAGATTTTGACATTTTGGAGTATTTAATCGAGCCTTTGTCAAAAATTCCAAACATATAT TTGCACCAAATTGACAAGΆATCTAΆTAAATTTGTCACGTAATCTTTTTGGAAGAATGTAT GAAAAGATCAACTACGACGCCAGCCAAACTCAAAAGGTTTTTAACAAAGAATACTTTACT ATGAATACGGTTACGCAACTCCCAACTTTTTTCATGTTTAAAGATGGTGATCCCATATCC TATGTTTTCCCCGGATACTCCACAACAGAAATGAGAAATATTGATGCCATTATGGATTGG GTAAAAAAGTATTCTAATCCCTTAGTTACCGAAGTTGACTCTTCTAATTTGAAAAAATTA ATTTCCTTCCAAACCAAGAGCTACAGTGATTTAGCAATTCAGTTAATAAGTAGCACTGAC CACAΆACATATCAAΆGGAAGCAACAAGCTTATTAAAAACTTGCTCCTCGCAAGTTGGGAG TATGAACATATTCGGATGGAAAATAACTTCGAAGAAATTAATGAGAGAAGGGCAAGGAAA GCAGACGGGATCAAGAAAATAAAGGAAΆΆAAAGGCTCCGGCTAACAAΆATTGTTGATAAA ATGCGTGAAGAGATTCCCCATATGGATCAAAAAAAATTGTTATTAGGATATTTAGATATT TCAAAGGAGAAGAATTTTTTTAGAAAATATGGTATTACTGGAGAATATAΆAATTGGTGAT GTGATTATCATTGATAAATCAAATAATTACTACTACAATAAAGATAATTTTGGCAACTCC TTGACTTCTAACAACCCTCAATTGCTGAGAGAAGCATTCGTGTCCTTAAATATTCCATCA AAAGCTCTATACAGCTCTAAGTTGAAGGGGAGATTGATAAATTCTCCATTCCATAΆTGTC CTCAGTTTCCTAGACATAATCCACGGGAACGGCATGCCCGGTTACTTAATTGTTATTGTT TTGTTTATCGCAATACTCAAAGGTCCATCTATTTACAGAAGATACAAAGTAAGGAAACAC TATAGGGCGAAAAGGAACGCTGTCGGTATCCTAGGAAATATGGAGAAAAAAAAAAATCAΆ
GATTAA
Further information on EPSl can be obtained from the URL address ■ http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000001267. It will be appreciated that, by "EPSl", we include fragments or variants thereof having equivalent EPSl -like activity.
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), is another S. cerevisiae helper protein of interest for the present invention. By "PDI" we include any protein having the ability to reactivate the ribonuclease activity against RNA of scrambled ribonuclease as described in EP 0 746 611 and Hillson et al, 1984, Methods Enzymol, 107, 281-292.
PDI is an enzyme which typically catalyses thiobdisulphide 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, ScL, 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 isomerisation, 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-
511). Recently, sulphydryl oxidation, not disulphide isomerisation was reported to be the principal function of Protein Disulphide Isomerase in S. 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, Biochemistiy, 28, 7298-7305; Lundstrom and Holmgren, 1990, J Biol. Chem., 265, 9114-9120; Hawkins and Freedman, 199O5 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 IcD) with characteristically acidic pi (4.0-4.5) (Hillson et 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 et 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 al, 1985, op. cit.).
Preferred PDI sequences include those from humans and those from yeast species, such as S. cerevisiae.
A yeast protein disulphide isomerase precursor, PDIl, can be found as Genbank accession no. CAA42373 or BAA00723, It has. the following sequence of 522 ammo acids:
1 mkfsagavls wsslllassv faqqeavape dsavvklatd sfneyigshd Ivlaeffapw
61 cghcknmape yvkaaetlve knitlaqidc tenqdlcmeh nipgfpslki fknsdvnnsi
121 dyegprtaea ivqfmikqsq pavavvadlp aylanetfvt pvivqsgkid adfnatfysm
181 ankhfndydf vsaenadddf klsiylpsam depvvyngkk adiadadvfe kwlqvealpy 241 fgeidgsvfa qyvesglplg ylfyndeeel eeykplftel akknrglmnf vsidarkfgr
301 hagnlnmkeq fplfaihdmt edlkyglpgl seeafdelsd kivleskaie slvkdflkgd
361 aspivksqei fenqdssvfq lvgknhdeiv ndpkkdvlvl yyapwcghck rlaptyqela
421 dtyanatsdv liakldhten dvrgvviegy ptivlypggk ksesvvyqgs rsldslfdfi 481 kenghfdvdg kalyeeaqek aaeeadadae ladeedaihd el
An alternative yeast protein disulphide isomerase sequence can be found as Genbank accession no. CAA38402. It has the following sequence of 530 amino acids
1 mkfsagavls wsslllassv faqqeavape dsawklatd sfneyiqshd lvlaeffapw
61 cghcknmape yvkaaetlve knitlaqidc tenqdlcmeh nipgfpslki fknrdvnnsi
121 dyegprtaea ivqfmikqsq pavavvadlp aylanetfvt pvivqsgkid adfnatfysm
161 ankhfndydf vsaenadddf klsiylpsam depvvyngkk adiadadvfe kwlqvealpy 241 fgeidgsvfa qyvesglplg ylfyndeeel eeykplftel akknrglmnf vsidarkfgr
301 hagnlnmkeq fplfaihdmt edlkyglpql seeafdelsd kivleskaie slvkdflkgd
361 aspivksqei fenqdssvfq Ivgknhdeiv ndpkkdvlvl yyapwcghck rlaptyqela
421 dtyanatsdv liakldhten dvrgvviegy ptivlypggk ksesvvyqgs rsldslfdfi
481 kenghfdvdg kalyeeaqek aaeeaeadae aeadadaela deedaihdel
The following alignment of these sequences (the sequence of Genbank accession no. CAA42373 or BAA00723 first, the sequence of Genbank accession no. CAA38402 second) 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.
1 mkfsagavls wsslllassv faqqeavape dsawklatd sfneyiqshd lvlaeffapw
1 mkfsagavls wsslllassv faqqeavape dsawklatd sfneyiqshd lvlaeffapw
61 cghcknmape yvkaaetlve knitlaqidc tenqdlcmeh nipgfpslki fknsdvnnsi
61 cghcknmape yvkaaetlve knitlaqidc tenqdlcmeh nipgfpslki fknrdvnnsi
121 dyegprtaea ivqfmikqsq pavavvadlp aylanetfvt pvivqsgkid adfnatfysm 181 dyegprtaea ivqfmikqsq pavavvadlp aylanetfvt pvivqsgkid adfnatfysm
181 ankhfndydf vsaenadddf klsiylpsam depvvyngkk adiadadvfe kwlqvealpy 181 ankhfndydf vsaenadddf klsiylpsam depvvyngkk adiadadvfe kwlqvealpy 241 fgeidgsvfa qyvesglplg ylfyndeeel eeykplftel akknrglmnf vsidarkfgr 241 fgeidgsvfa qyvesglplg ylfyndeeel eeykplftel akknrglmnf vsidarkfgr
301 hagnlnmkeq fplfaihdmt edlkyglpql seeafdelsd kivleskaie slvkdflkgd 301 hagnlnmkeq fplfaihdmt edlkyglpql seeafdelsd kivleskaie 'slvkdflkgd
361 aspivksqei fenqdssvfq Ivgknhdeiv ndpkkdvlvl yyapwcghck rlaptyqela 361 aspivksqei fenqdssvfq Ivgknhdeiv ndpkkdvlvl yyapwcghck rlaptyqela
421 dtyanatsdv liakldhten dvrgvviegy ptivlypggk ksesvvyqgs rsldslfdfi 421 dtyanatsdv liakldhten dvrgvviegy ptivlypggk ksesvvyqgs rsldslfdfi
481 kenghfdvdg kalyeeaqek aaeea***** ***dadaela deedaihdel 481 kenghfdvdg kalyeeaqek aaeeaeadae aeadadaela deedaihdel
It will be appreciated that, by "PDI" and "PDIl", we include fragments or variants thereof having equivalent PDI-like activity and PDIl -like activity, respectively.
DERI is another S. cerevisiae helper protein of interest for the present invention. Derlp is an endoplasmic reticulum membrane protein, required for the protein degradation process associated with the ER, and is involved in the retrograde transport of misfolded or unassembled proteins. A published protein sequence for the protein Derlp is as follows:
MDAVILNLLGDIPLVTRLWTIGCLVLSGLTSLRIVDPGKWYSYDLVFKKGQYGRLLYSI FDYGAFNWISMINIFVSANHLSTLENSFNLRRKFCWIIFLLLVILVKMTSIEQPAASLGV LLHENLVYYELKKNGNQMNVRFFGAIDVSPSIFPIYMNAVMYFVYKRSWLEIAMNFMPGH VIYYMDDIIGKIYGIDLCKSPYDWFRNTETP*
DERI is encoded by a non-essential gene comprising an ORF that is 0.636 kbp in size and is located on chromosome II. A published nucleotide coding sequence of DERI is as follows, 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:
ATGGATGCTGTAATACTGAATCTCTTAGGCGACATTCCTTTGGTCACAAGATTATGGACA ATTGGCTGTCTTGTACTATCAGGTCTCACAAGTCTCCGGATTGTGGATCCAGGGAAGGTA GTGTACAGTTATGATTTAGTATTCAAAAAGGGACAATATGGAAGACTACTTTATTCGATA TTCGATTACGGCGCATTTAATTGGATATCCATGATAAACATCTTTGTCAGCGCTAATCAC TTATCAACTTTGGAAAACTCATTCAATCTGAGAAGAAAATTCTGTTGGATAATATTTTTA
CTGTTGGTGATACTGGTAAΆGATGACCAGCATTGAACAACCTGCAGCATCACTCGGTGTG
TTATTGCATGAGAATCTCGTGTACTACGAACTGAAAAAGAACGGAAACCAAATGAACGTA
• CGATTCTTCGGTGCCATTGATGTTTCACCATCTATATTCCCAATCTACATGAATGCAGTA
ATGTATTTTGTATATAAGCGTAGCTGGTTAGAAATTGCCATGAATTTCATGCCAGGTCAC GTAATTTACTACATGGATGATATAATAGGGAAGATTTATGGCATCGATTTGTGTAAATCT CCGTACGACTGGTTCCGCAACACTGAAACACCCTAA Further information on DERI can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000000405.
It will be appreciated that, by "DERI", we include fragments or variants thereof having equivalent DERI -like activity.
DER3 is another S. cerevisiae helper protein of interest for the present invention and is also known as HRDl. Der3p is a ubiquitin-protein ligase required for endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins. It is genetically linked to the unfolded protein response (UPR) and is thought to be regulated through association with Hrd3p. It contains an H2 ring finger. A published protein sequence for the protein Der3p is as follows:
MVPENRRKQLAIFVWTYLLTFYCVYSATKTSVSFLQVTLKLNEGFNLMVLSIFILLNST LLWQLLTKLLFGELRLIEHEHIFERLPFTIINTLFMSSLFHERYFFTVAFFGLLLLYLKV FHWILKDRLEALLQSINDSTTMKTLIFSRFSFNLVLLAVVDYQIITRCISSIYTNQKSDI ESTSLYLIQVMEFTMLLIDLLNLFLQTCLNFWEFYRSQQSLSNENNHIVHGDPTDENTVE SDQSQPVLNDDDDDDDDDRQFTGLEGKFMYEKAIDVFTRFLKTALHLSMLIPFRMPMMLL KDVVWDILALYQSGTSLWKIWRNNKQLDDTLVTVTVEQLQNSANDDNICIICMDELIHSP NQQTWKNKNKKPKRLPCGHILHLSCLKNWMERSQTCPICRLPVFDEKGNWQTTFTSNSD ITTQTTVTDSTGIATDQQGFANEVDLLPTRTTSPDIRIVPTQNIDTLAMRTRSTSTPSPT WYTFPLHKTGDNSVGSSRSAYEFLITNSDEKENGIPVKLTIENHEVNSLHGDGGEQIAKK IVIPDKFIQHI*
DER3 is encoded by a non-essential gene comprising an ORF that is 1.656 kbp in size and is located on chromosome XV. A published nucleotide coding sequence of DER3 is as follows, 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:
ATGGTGCCAGAAAATAGAAGGAAACAGTTGGCAATTTTTGTAGTTGTCACATATTTGCTC ACATTTTATTGCGTGTATTCAGCCACCAAGACAAGCGTTTCCTTTTTGCAAGTAACACTG AAGCTAAATGAAGGCTTCAATCTAATGGTTTTGTCGATATTCATCTTATTAAATTCTACC TTACTATGGCAACTCCTAACGAAACTATTATTTGGTGAACTGAGGCTTATTGAGCATGAG CACATTTTTGAAAGGTTACCATTTACCATTATAAACACCTTGTTTATGTCCTCACTGTTC CACGAACGGTATTTTTTCACAGTGGCATTTTTTGGACTATTACTACTCTATCTGAAAGTT TTCCATTGGATTTTAAAGGATAGGCTGGAGGCCTTATTACAGTCAATAAATGATTCCACC ACAATGAΆΆACCCTTATCTTTAGTAGATTCTCATTTAACCTCGTACTATTGGCGGTTGTA GACTACCAGATAATAACACGATGCATCTCCTCCATATATACAΆACCAAΆAGAGTGATATT GAATCCACATCCCTTTACCTGATACAAGTAATGGAGTTTACCATGCTTTTGATTGATTTG CTAAATTTATTCCTACAGACTTGTTTGAATTTCTGGGAATTTTATCGCTCACAACAAAGT CTGTCTAATGAGAACAACCATATTGTCCATGGCGATCCTACAGATGΆAAACACGGTTGAG TCTGATCAΆTCTCAGCCAGTGCTGAATGACGACGACGATGACGACGATGATGATAGACAΆ TTTACCGGCCTGGAGGGTAAATTCATGTATGAAAAAGCAATTGACGTATTCACAAGATTC TTAAAAACGGCACTTCATTTGTCTATGCTAATACCATTTAGGATGCCTATGATGCTTTTG AAΆGATGTGGTGTGGGATATCTTGGCACTATATCAAAGTGGCACAΆGTTTGTGGAAAΆTC TGGAGAAATAACAAACAGCTCGACGACACTCTTGTCACTGTCACCGTAGAACAGCTACAA AATTCTGCAAATGATGACAATATTTGTATCATTTGTATGGATGAGTTAATACATTCTCCA AACCAGCAGACGTGGAAGAATAAAAΆCAAGAAACCCAAAAGGTTACCTTGTGGCCACATA CTTCATTTGTCGTGTTTAAAGAATTGGATGGAACGTTCTCAGACTTGTCCTATTTGTAGA TTGCCTGTCTTTGATGAAΆAAGGTAΆTGTTGTGCAAACGACTTTCACTTCCAATAGTGAT ATCACGACACAGACCACCGTAACAGATAGCACTGGGATAGCGACAGATCAACAAGGTTTC GCAAACGAAGTAGATCTACTTCCCACAAGAACAACTTCCCCTGATATAAGGATAGTGCCT ACTCAAAATATAGACACATTAGCAATGAGAACAAGGTCAACCTCTACACCATCTCCTACG TGGTATACGTTCCCATTACATAAAACTGGTGATAATTCTGTTGGGTCAAGCCGATCAGCC TACGAATTTTTGATCACAAATTCAGATGAGAAAGAAAATGGTATTCCTGTCAAATTAACA ATAGAΆAATCACGAAGTAAATTCTCTGCATGGAGACGGGGGCGAGCAAATTGCCAAGAΆA ATTGTCATACCAGATAAΆTTTATCCAGCATATCTAG
Further information on DER3 can be obtained from the URL address http://db.yeastgenome.org/cgi-bm/singlepageformat?sgdid=::S000005373.
It will be appreciated that, by "DER3", we include fragments or variants thereof having equivalentDER3-like activity.
HRD3 is another S. cerevisiae helper protein ofinterest for the present invention.
Hrd3p is a resident protein ofthe ER membrane that plays a central role in ER- associated protein degradation (ERAD). It forms an HRD complex with Hrdlp and ERAD determinants that engage in lumen to cytosol communication and coordination of ERAD events. A published protein sequence for the protein
Hrd3p is asfollows: MITLLLYLCVICNAIVLIRADSIADPWPEARHLLNTIAKSRDPMKEAAMEPNADEFVGFY VPMDYSPRNEEKNYQSIWQNEITDSQRHIYELLVQSSEQFNNSEATYTLSQIHLWSQYNF PHNMTLAHKYLEKFNDLTHFTNHSAIFDLAVMYATGGCASGNDQTVIPQDSAKALLYYQR AAQLGNLKAKQVLAYKYYSGFNVPRNFHKSLVLYRDIAEQLRKSYSRDEWDIVFPYWESY , NVRISDFESGLLGKGLNSVPSSTVRKRTTRPDIGSPFIAQVNGVQMTLQIEPMGRFAFNG NDGNINGDEDDEDASERRIIRIYYAALNDYKGTYSQSRNCERAKNLLELTYKEFQPHVDN LDPLQVFYYVRCLQLLGHMYFTGEGSSKPNIHMAEEILTTSLEISRRAQGPIGRACIDLG LINQYITNNISQAISYYMKAMKTQANNGIVEFQLSKLATSFPEEKIGDPFNLMETAYLNG FIPAIYEFAVMIESGMNSKSSVENTAYLFKTFVDKNEAIMAPKLRTAFAALINDRSEVAL WAYSQLAEQGYETAQVSAAYLMYQLPYEFEDPPRTTDQRKTLAISYYTRAFKQGNIDAGV VAGDIYFQMQNYSKAMALYQGAALKYSIQAIWNLGYMHEHGLGVNRDFHLAKRYYDQVSE HDHRFYLASKLSVLKLHLKSWLTWITREKVNYWKPSSPLNPNEDTQHSKTSWYKQLTKIL QRMRHKEDSDKΆAEDSHKHRTVVQNGANHRGDDQEEASEILGFQMEDLVTMGCILGIFLL SILMSTLAARRGWNVRFNGAQLNANGNRQQEQQQQQQAQGPPGWDFNVQIFAI*
HRD3 is encoded by anon-essential gene comprising an ORF that is 2.502 kbp in size and is located on chromosome XII. A published nucleotide coding sequence ofHKD3 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGATAACACTCTTATTATACCTGTGCGTAATATGTAACGCAATAGTGTTAATAAGGGCT GATTCGATAGCGGACCCTTGGCCTGAAGCGCGACATCTACTAAATACCATAGCTAAGTCC AGAGACCCAATGAAΆGAΆGCTGCTATGGAΆCCCAATGCAGATGAATTTGTTGGATTCTAT GTACCGATGGATTATTCCCCACGTAATGAGGAAAAAAACTACCAGAGCATTTGGCAAAAC GAAATCACAGATTCTCAACGTCATATTTATGAΆTTACTTGTACAATCAΆGTGAACAATTC AACAACTCAGAAGCAACATATACACTTAGCCAGATTCACCTTTGGAGTCAATATAATTTC CCGCATAATATGACTTTGGCACACAAATACTTAGAAAAATTCAATGATCTAACCCACTTC ACCAATCATTCGGCCATCTTCGACTTAGCTGTGATGTATGCCACTGGGGGATGTGCTTCT GGTAATGATCAAACCGTGATCCCTCAGGATTCTGCTAAAGCACTGCTATATTACCAAAGG GCTGCCCAACTAGGGAATTTAAAGGCTAAGCAAGTGCTAGCTTATAAATACTATTCTGGC TTCAATGTCCCACGAAATTTTCATAAATCTTTAGTATTGTACAGGGACATTGCTGAACAG CTGAGAAAGTCGTACTCCAGGGACGAATGGGATATTGTCTTCCCCTATTGGGAAAGTTAC ΆACGTGAGAATATCGGATTTTGAGAGTGGCCTATTAGGTAAAGGTTTGAΆTTCCGTTCCA TCTTCTACAGTAAGGAAAAGAACTACGAGACCAGATATTGGTTCACCCTTTATTGCGCAA GTTAACGGTGTACAGATGACCTTGCAAATCGAACCGATGGGTAGGTTCGCTTTCAACGGT AACGATGGCAACATAAATGGCGACGAAGATGACGAGGATGCCAGTGAAAGACGAATCATT CGGATATATTATGCAGCTTTGAATGATTATAAAGGAACATATTCACAAAGCAGAAATTGT GAGCGCGCCAAAAACTTGTTGGAATTAACGTACAAGGAATTTCAGCCTCATGTCGACAAT TTGGATCCTTTGCAAGTATTTTACTACGTCCGTTGCTTACAATTATTGGGGCACATGTAT
TTCACCGGCGAAGGCTCCTCGAAGCCTAATATTCATATGGCCGAΆGAGATCCTGACCACG TCGCTAGAAATAAGCAGAAGGGCACAGGGACCTATAGGTAGAGCGTGCATAGATCTGGGC TTAATAAATCAATACATCACAAACAATATTTCTCAAGCAATTTCGTATTATATGAAAGCT ATGAAAACACAAGCTAACAATGGAATCGTAGAATTCCAATTATCCAAATTGGCCACTTCA TTCCCTGAAGAAAAAATCGGCGACCCATTTAACTTAATGGAAACTGCCTACTTGAATGGA TTCATTCCAGCCATATATGAGTTTGCAGTAATGATCGAATCTGGAATGAACAGTAAGAGT AGTGTGGAAAACACTGCTTACCTGTTCAAAΆCATTCGTTGACAAAAACGAAGCTATTATG GCACCTAAACTGAGGACAGCATTTGCCGCATTAATCAACGATCGTTCAGAAGTGGCTTTA TGGGCTTATTCCCAACTAGCCGAGCAAGGCTACGAGACTGCTCAAGTCTCTGCCGCCTAC TTAATGTACCAGTTGCCATATGAGTTTGAGGATCCTCCAAGAACCACAGATCAGAGAAAA ACTTTGGCAATTTCCTACTATACAAGAGCGTTTAAACAGGGAAATATAGATGCTGGTGTT GTCGCGGGAGATATCTATTTTCAGATGCAGAATTACAGTAAAGCTATGGCTCTTTATCAG GGTGCAGCTTTGAAGTACTCTATACAGGCTATCTGGAACTTAGGGTACATGCATGAGCAT GGGCTAGGTGTAAACAGAGATTTCCATCTTGCTAAΆCGTTACTACGACCAAGTTTCAGAA CACGATCATAGATTTTACTTGGCTTCCAAATTGAGTGTTTTAAΆATTACACCTAAAGTCA TGGTTGACTTGGATCACCAGAGAAAAAGTAAΆCTACTGGAAACCTTCCTCGCCACTTAAC CCTAACGAΆGATACTCAGCACTCGAΆGACTTCATGGTACAAGCAATTGACGAAGATTCTA CAAAGAATGAGACATAAGGAGGATAGTGACAAAGCTGCGGAAGATTCTCACAAACACAGA ACTGTAGTGCAGAATGGAGCTAACCATAGGGGTGACGACCAAGAGGAGGCTTCCGAGATT TTGGGCTTCCAAATGGAGGATCTTGTTACGATGGGATGTATCTTGGGGATATTCCTATTA AGTATATTAATGAGTACACTGGCGGCCCGTAGAGGCTGGAATGTCCGTTTCAATGGAGCA CAATTAΆATGCAAATGGTAACCGGCAGCAAGAGCAΆCAACAACAACAACAAGCACAAGGT CCCCCGGGCTGGGACTTCAATGTTCAGATATTCGCCATATGA
Further information on HRD3 can be obtained .from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000004197.
It will be appreciated that, by "HRD3", we include fragments or variants thereof having equivalentHRD3-like activity.
UBC7 is another S. cerevisiae helper protein ofinterest for the present invention andis also known as QRI8. Ubc7p is aubiquitin conjugating enzyme, involved in the ER-associated protein degradationpathway. It requires Cuelp for recruitment to the ER membrane and is proposed to be involved in chromatin assembly. A publishedprotein sequence fortheproteinUbc7p is as follows: MSKTAQKRLLKELQQLIKDSPPGIVAGPKSENNIFIWDCLIQGPPDTPYADGVFNAKLEF PKDYPLSPPKLTFTPSILHPNIYPNGEVCI SILHSPGDDPNMYELAEERWSPVQSVEKIL LSVMSMLSEPNIESGANIDACILWRDNRPEFERQVKLSILKSLGF*
UBC7 is encoded by anon-essential gene comprising an ORP that is 0.498 kbp in size and is located on chromosome XIII. A published nucleotide coding sequence of UBC7 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGTCGAAAACCGCTCAGAAACGTCTCCTCAAGGAGCTTCAACAGTTAATTAAAGATTCT CCACCTGGTATAGTGGCTGGTCCCAAATCGGAGAATAACATATTCATTTGGGACTGCCTA ΆTTCAAGGGCCTCCAGATACGCCATACGCTGATGGTGTTTTTAATGCTAAGCTAGAGTTT CCTAAAGACTATCCGTTATCTCCACCTAAACTTACTTTCACACCCAGCATACTACATCCA AΆTATTTATCCAAATGGGGAAGTGTGCΆTATCCATTCTACACTCCCCTGGTGATGATCCT AACATGTACGAΆTTAGCGGAΆGAAΆGATGGTCGCCAGTGCAAAGTGTAGAAAAΆATTCTA TTAAGTGTTATGAGCATGTTGAGTGAGCCCAATATCGAAAGTGGTGCCAACATTGATGCT TGCATCTTGTGGAGAGATAATAGACCTGAATTTGAGAGACAGGTAAAGTTATCCATTTTG AAATCATTAGGATTCTGA
Further information on UBC7 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000004624.
It will be appreciated that, by "UBC7", we include fragments or variants thereof having equivalent UBC7-like activity.
DOA4 is another S. cerevisiae helper protein of interest for the present invention and is also known as DOSl, MUT4, NPI2, SSV7, and UBP4. Doa4p is a ubiquitin hydrolase, required for recycling ubiquitin from proteasome-bound ubiquitinated intermediates, which, acts at the late endosome/prevacuolar compartment to recover ubiquitin from ubiquitinated membrane proteins en route to the vacuole. A published protein sequence for the protein Doa4p is as follows:
MEQNIISTIRDECIRHRSKYLTIAQLTAIAEAKINEFIITGKAKDQDLSSLLDKCIDILS IYKKNSKDIKNIISCKNKGAMISSNSVMIIQLNYVYYKVIHIIVTTNIPHLSEFAKIKLH KSTSDEGNGNNNNNEFQLMNIYNTLLETLLKDENIAKIKSFIKSSIKQTKLNHEQEECNL MRTGSYITSNQLNSLISSSANSASSQMEILLIDIRSRLEFNKSHIDTKNIICLEPISFKM SYSDHDLEKKSLITSPNSEIKMFQSR]SILFKFIILYTDANEYNVKQQSVLLDILVNHSFEK PISDDFTKIFILESGFPGWLKSNYGRQVSSSFPSNNNIKDDSVYINGNTSGLSLQHLPKM SPSIRHSMDDSMKEMLVAPTPLNHLQQQQQQQSDNDHVLKRSSSFKKLFSNYTSPNPKNS NSNLYSISSLSISSSPSPLPLHSPDPVKGNSLPINYPETPHLWKNSETDFMTNQREQLNH NSFAHIAPINTKAITSPSRTATPKLQRFPQTISMNLNMNSNGHSSATSTIQPSCLSLSNN DSLDHTDVTPTSSHNYDLDFAVGLENLGNSCYMNCIIQCILGTHELTQIFLDDSYAKHIN INSKLGSKGILAKYFARLVHMMYKEQVDGSKKISISPIKFKLACGSVNSLFKTASQQDCQ EFCQFLLDGLHEDLNQCGSNPPLKELSQEAEARREKLSLRIASSIEWERFLTTDFSVIVD LFQGQYASRLKCKVCSHTSTTYQPFTVLSIPIPKKNSRNNITIEDCFREFTKCENLEVDE QWLCPHCEKRQPSTKQLTITRLPRNLIVHLKRFDNLLNKNNDFVIYPFLLDLTPFWANDF DGVFPPGVNDDELPIRGQIPPFKYELYGVACHFGTLYGGHYTAYVKKGLKKGWLYFDDTK YKPVKNKADAINSNAYVLFYHRVYGV*
DOA4 is encoded by anon-essential gene comprising an ORF that is 2.781 kbp in size and is located on chromosome IV. A published nucleotide coding sequence ofDOA4 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGGAGCAGAATATTATTAGTACCATAAGGGATGAGTGTATTCGTCACCGGTCGAAGTAC CTTACGATAGCACAACTAACCGCTATTGCAGAGGCTAAAATTAACGAATTCATCATAACT GGTAAGGCAAAAGATCAAGATTTGAGCAGTCTTCTAGATAAATGCATCGATATTTTATCT ATTTACAAGAAGAACTCGAΆAGATATCAAAAATATTATATCGTGCAAAAATAAGGGTGCA ATGATTAGTTCAAATTCCGTAATGATTATTCAATTAAATTATGTTTACTACAΆGGTAATT CACATTATTGTAACAACCAATATTCCTCATTTAAGTGAATTCGCCAAGATTAAATTACAT AAGAGCACGAGTGATGAGGGCAACGGTAATAACAACAATAATGAATTTCAACTCATGAAC ATTTACAACACTTTGCTGGAAACCTTATTAAAAGATGAAAACATTGCAAAAATAAAAAGT TTCATTAAGTCTTCCATAAAACAAACAAAATTGAACCATGAGCAAGAAGAATGTAACCTG ATGAGAACGGGTTCCTATATCACTTCCAATCAATTAAACTCCCTAATAAGTTCATCAGCA AATTCTGCTTCCTCCCAAΆTGGAGATACTACTGATAGATATACGATCAAGGTTGGAATTC AACAAGTCACATATTGATACAAAAAATATTATATGCCTGGAGCCTATTTCTTTTAAAATG TCATATTCAGATCATGATTTGGAGAAAAAATCATTAATTACTTCTCCTAATAGTGAGATT AAAATGTTTCAAAGTAGAAATCTTTTCAAGTTTATCATTCTCTATACAGACGCAAACGAA TACAATGTTAAACAGCAGTCTGTCCTGTTGGACATTCTGGTGAATCATTCCTTTGAΆAAA CCAATATCCGATGACTTTACCAAAATTTTCATTCTGGAATCTGGTTTTCCAGGTTGGCTT AΆGTCAAΆTTATGGGAGGCAAGTATCATCATCTTTTCCATCAAATAACAATATTAAAGAT GATAGTGTTTATATTAΆTGGTAΆCACTTCTGGCCTAΆGTTTACAACATTTACCTAAGATG TCTCCCAGTATAAGACATTCAATGGACGACTCTATGAAAGAAΆTGCTAGTTGCGCCTACT CCATTAAATCATCTTCAACAΆCAGCAACAACAGCAATCAGACAATGATCATGTGCTAAAA AGATCTTCAAGTTTCAAAAAATTATTCTCAAATTATACGTCTCCTAATCCGAAGAATTCA AATTCAAACTTATATTCTATATCTTCGTTGTCCATATCTAGTTCACCΆTCGCCTTTACCT CTACATTCGCCTGACCCAGTTAAGGGCAATTCATTGCCAATCAATTATCCGGAAACGCCA CATCTTTGGAΆΆAΆCAGTGAGACAGATTTTATGACAAATCAAAGAGAΆCAGTTGAATCAC AACTCTTTTGCTCACATAGCTCCTATCAACACGAAGGCCATCACTTCTCCATCAAGAACT GCCACACCGAAGTTACAACGCTTCCCGCAAACAATTAGTATGAACCTTAATATGAACTCC AATGGACACAGTTCTGCCACCTCTACCATTCAACCTTCGTGTCTATCCTTGTCTAATAAT GACTCTTTAGATCATACAGATGTTACACCAACTTCTTCTCATAΆTTATGACCTTGATTTC GCGGTTGGTTTGGAAΆATCTAGGAAATTCGTGTTACATGAACTGTATCATTCAGTGTATC TTAGGTACACACGAATTAACCCAAATCTTTTTGGACGATTCATATGCTAAΆCACATCAAT ATTAATAGTAAGTTGGGATCGAAAGGTATTCTGGCAAAATATTTTGCAAGGTTGGTTCAT ATGATGTATAAGGAACAGGTTGATGGTTCAAAGAAAATTTCCATATCACCGATAΆΆATTT AΆATTGGCATGTGGATCTGTTAACTCATTATTTAAGACTGCATCCCAΆCAGGACTGCCAA GAGTTTTGCCAΆTTCCTTCTAGATGGTCTTCATGAΆGACTTGAACCAATGCGGTTCAAAC CCACCTTTGAΆGGAGCTTTCTCAAGAAGCTGAGGCGAGAAGAGAAAAACTGTCTTTGCGΆ ATTGCCTCGTCAATTGAGTGGGAACGATTCTTGACTACTGATTTCAGTGTTATTGTCGAC TTATTTCAGGGACAATACGCCTCACGACTAAAATGTAAAGTCTGTAGTCATACCTCGACA ACATACCAACCTTTTACGGTGCTGTCAATCCCTATTCCTAAAAΆAΆATTCCCGAAATAAT ATTACCATTGAAGATTGTTTCAGAGAGTTCACCAAATGTGAGAACTTGGAAGTGGATGAG CAATGGTTGTGCCCACATTGTGAAAΆΆAGGCAGCCCTCCACGAAACAATTGACAATAACG AGATTACCGAGGAATCTGATAGTCCATTTAAAGAGATTTGATAATTTATTAAACAAAAAT AATGACTTCGTCATATACCCTTTTTTGTTGGACTTGACTCCATTTTGGGCCAATGATTTT GACGGGGTTTTTCCTCCAGGTGTTAATGACGATGAACTACCAATAAGGGGACAAATACCA CCTTTTAAGTATGAATTATATGGTGTAGCATGCCACTTTGGTACTTTGTATGGTGGTCAT TATACAGCCTATGTGAAAAAGGGATTAAAGAAGGGATGGCTATATTTTGATGATACCAAA TATAAACCTGTCAAAAACAAAGCCGATGCAATTAACTCTAATGCATACGTTTTGTTTTAT CACCGCGTCTACGGTGTTTGA
Further information ■ on DOA4 can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S000002476.
It will be appreciated that, by "DOA4", we include fragments or variants thereof having equivalentDOA4-like activity. HACl is another S. cerevisiae helper protein of interest for the present invention, and is also known as ERN4 and IRE15. Haclp, is a bZIP transcription factor (ATF/CREB1 homolog) that regulates the unfolded protein response, via UPRE binding, and membrane biogenesis. ER stress-induced splicing pathway utilising Irelp, Trllp and Ada5p facilitates efficient Haclp synthesis. A published protein sequence for the protein Haclp is as follows:
MEMTDFELTSNSQSNLAIPTNFKSTLPPRKRAKTKEEKEQRRIERILRNRRAAHQSREKK RLHLQYLERKCSLLENLLNSVNLEKLADHEDALTCSHDAFVASLDEYRDFQSTRGASLDT RASSHSSSDTFTPSPLNCTMEPATLSPKSMRDSASDQETSWELQMFKTENVPESTTLPAV DNNNLFDAVASPLADPLCDDIAGNSLPFDNSIDLDNWRNPEAQSGLNSFELNDFFITS*
HACl is encoded by a non-essential gene that is located on chromosome VI. A published nucleotide coding sequence of HACl, that has been processed to remove introns, is 0.717 kbp in size and is as follows (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):
ATGGAAATGACTGATTTTGAACTAACTAGTAATTCGCAATCGAACTTGGCTATCCCTACC AACTTCAAGTCGACTCTGCCTCCAAGGAAAAGAGCCAAGACAΆAAGAGGAAAAGGAACAG CGAAGGATCGAGCGTATTTTGAGAAACAGAAGAGCTGCTCACCAGAGCAGAGAGAAAAAA AGACTACATCTGCAGTATCTCGAGAGAAΆΆTGTTCTCTTTTGGAAAΆTTTACTGAΆCAGC GTCAACCTTGAAAAACTGGCTGACCACGAΆGACGCGTTGACTTGCAGCCACGACGCTTTT GTTGCTTCTCTTGACGAGTACAGGGATTTCCAGAGCACGAGGGGCGCTTCACTGGACACC AGGGCCAGTTCGCACTCGTCGTCTGATACGTTCACACCTTCACCTCTGΆΆCTGTACAΆTG GAGCCTGCGACTTTGTCGCCCAAGAGTATGCGCGATTCCGCGTCGGACCAAGAGACTTCA TGGGAGCTGCAGATGTTTAAGACGGAAAATGTACCAGAGTCGACGACGCTACCTGCCGTA GACAΆCAACAATTTGTTTGATGCGGTGGCCTCGCCGTTGGCAGΆCCCACTCTGCGACGAT ATAGCGGGAAACAGTCTACCCTTTGACAATTCAATTGATCTTGACAATTGGCGTAATCCA GAAGCGCAGTCAGGTTTGAATTCATTTGAATTGAATGATTTCTTCATCACTTCATGA
Further information on HACl can be obtained from the URL address http://db.yeastgenome.org/cgi~bin/smglepageformat?sgdid=S000001863. It will be appreciated that, by "HACl", we include fragments or variants thereof having equivalent HACl -like activity.
SEC63 is another S. cerevisiae helper protein of interest for the present invention. It is also known as PTLl. It is an essential subunit of the Sec63 complex (Sec63p, Sec62p, Sec66p and Sec72p); with Secόl complex, Kar2p/BiP and Lhslp it forms a channel competent for SRP-dependent and post-translational SRP -independent protein targeting and import into the ER. A published protein sequence for the protein Sec63p is as follows:
MPTNYEYDEASETWPSFILTGLLMWGPMTLLQIYQIFFGANAEDGNSGKSKEFNEEVFK NLNEEYTSDEIKQFRRKFDKNSNKKSKIWSRRNIIIIVGWILVAILLQRINSNDAIKDAA TKLFDPYEILGISTSASDRDIKSAYRKLSVKFHPDKLAKGLTPDEKSVMEETYVQITKAY ESLTDELVRQNYLKYGHPDGPQSTSHGIALPRFLVDGSASPLLVVCYVALLGLILPYFVS RWWARTQSYTKKGIHNVTASNFVSNLVNYKPSEIVTTDLILHWLSFAHEFKQFFPDLQPT DFEKLLQDHINRRDSGKLNNAKFRIVAKCHSLLHGLLDIACGFRNLDIALGAINTFKCIV QAVPLTPNCQILQLPNVDKEHFITKTGDIHTLGKLFTLEDAKIGEVLGIKDQAKLNETLR VASHIPNLKIIKADFLVPGENQVTPSSTPYISLKVLVRSAKQPLIPTSLIPEENLTEPQD FESQRDPFAMMSKQPLVPYSFAPFFPTKRRGSWCCLVSSQKDGKILQTPIIIEKLSYKNL NDDKDFFDKRIKMDLTKHEKFDINDWEIGTIKIPLGQPAPETVGDFFFRVIVKSTDYFTT DLDITMNMKVRDSPAVEQVEVYSEEDDEYSTDDDETESDDESDASDYTDIDTDTEAEDDE
SPE*
SEC63 is encoded by an essential gene comprising an ORF that is 1.192 kbp in size and is located on chromosome XV. A published nucleotide coding sequence of SEC63 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
ATGCCTACAAATTACGAGTATGATGAGGCTAGTGAGACGTGGCCGTCCTTCATTTTAACG GGGCTCTTGATGGTCGTCGGGCCTATGACACTGCTTCAAATATACCAAΆTTTTTTTTGGG GCCAATGCTGAAGATGGGAATTCAGGGAAGAGTAAGGAGTTTAATGAGGAAGTTTTCAAG AACTTGAATGAAGAATACACCAGTGATGAAATCAAACAATTTAGAAGGAAGTTTGATAAA AATAGTAATAAGAAGTCCAAAATATGGAGCAGGAGAAATATTATAATTATTGTGGGTTGG ATCTTAGTTGCAATTCTTCTGCAAAGGATTAATAGTAATGACGCGATTAAAGACGCTGCT ACAAΆATTATTTGATCCTTATGAAATCCTTGGTATCTCTACTAGTGCTTCCGATAGAGAC ATCAAATCTGCTTATAGAAAATTATCTGTTAAATTTCATCCAGATAAATTAGCAAAGGGC CTAACACCTGATGAGAAAΆGTGTGATGGAAGAAACTTATGTTCAGATTACGAAGGCTTAC GAATCCCTTACTGACGAATTGGTTAGGCAΆΆΆCTATTTGAAATACGGTCATCCAGATGGC CCACAATCTACTTCACATGGTATCGCTCTACCAAGATTTTTGGTAGATGGAAGTGCATCT CCATTATTAGTGGTTTGTTATGTTGCGCTACTAGGTTTAATCTTGCCATATTTTGTTAGT AGATGGTGGGCAAGAACACAATCGTATACTAAGAAGGGAATACATAATGTGACGGCTTCT AΆTTTTGTTAGTAΆCTTAGTCAATTACAAGCCATCTGAGATTGTCACCACAGATTTGATC TTACACTGGTTATCATTTGCTCATGAATTTAAACAATTCTTCCCGGATTTGCAACCAACG GATTTTGAΆΆAACTTTTGCAAGATCATATTAΆCCGCAGAGATAGTGGTAAΆCTTAΆCAAT GCGAAATTTAGAATAGTGGCCAAATGTCACTCTTTGTTACACGGTTTATTGGATATTGCT TGTGGATTCAGAAATTTAGATATTGCATTGGGTGCAATCAATACTTTCAAGTGTATTGTT CAGGCTGTACCATTAACACCAAACTGTCAAΆTCCTTCAATTGCCGAACGTAGATAAΆGAG CACTTTATTACCAAAACCGGAGATATTCATACATTAGGTAΆATTGTTTACTTTAGAΆGAT GCCAΆGATTGGTGAGGTTCTTGGAATΆΆAGGATCAAGCAAΆGTTAAACGAAACTTTGAGA GTTGCATCGCATATTCCAAATCTAAAGATCATCAAGGCAGACTTCCTTGTCCCAGGTGAG AΆCCAAGTAACACCATCATCTACCCCATACATTTCTTTGAAΆGTACTGGTTCGTTCTGCT AΆACAGCCATTGATACCAΆCTAGCTTAATTCCTGAAGAAAATTTAACAGAACCTCAAGAT TTTGAΆTCTCAAAGAGATCCATTTGCTATGATGAGTAAACΆGCCACTCGTCCCATATTCC TTTGCACCATTTTTCCCTACAAAGAGACGTGGGAGTTGGTGCTGTCTGGTAAGTTCTCAA AAAGATGGTAAAATACTTCAAΆCGCCAATTATCATTGAAΆAGCTATCTTACAAGAΆCTTG AACGATGACAΆAGATTTCTTTGATAAGAGGATAAAAΆTGGATTTAACCAAACΆCGAΆΆAA TTCGATATAAATGATTGGGAAΆTCGGGACCATAAAAATTCCATTAGGTCAGCCTGCACCT GAΆACTGTTGGTGATTTCTTTTTTAGAGTAATCGTTAAATCCACAGATTATTTCACTACA GATTTGGATATTACCATGAATATGAAAGTTCGTGATTCTCCTGCAGTGGAACAAGTAGAG GTGTATTCTGAGGAGGATGATGAGTACTCTACTGATGACGACGAAACCGAAAGTGATGAT GAAAGTGATGCTAGCGΆTTATACTGATATCGATACGGATACAGAAGCTGAAGATGATGAA TCACCAGAΆTAG
Furtherinformation on SEC63 canbe obtainedfromtheURL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=S00000578Q
It will be appreciated that, by "SEC63", we include fragments or variants thereof having equivalent SEC63-like activity.
YDJl is another S. cerevisiae helper protein ofinterest for the present invention. It is also known as MAS5 and HSP40. It is a protein chaperone involved in regulation ofthe HSP90 and HSP70 functions; involved in protein translocation across membranes; member ofthe DnaJ family, and is located in the cytoplasm. Apublished protein sequence fortheprotein Ydjlp is as follows:
MVKETKFYDILGVPVTATDVEIKKAYRKCALKYHPDKNPSEEAAEKFKEASAAYEILSDP EKRDIYDQFGEDGLSGAGGAGGFPGGGFGFGDDIFSQFFGAGGAQRPRGPQRGKDIKHEI
SASLEELYKGRTAKLALNKQILCKECEGRGGKKGAVKKCTSCNGQGIKFVTRQMGPMIQR
FQTECDVCHGTGDIIDPKDRCKSCNGKKVENERKILEVHVEPGMKDGQRIVFKGEADQAP
DVIPGDVVFIVSERPHKSFKRDGDDLVYEAEIDLLTAIAGGEFALE-HVSGDWLKVGIVPG EVIAPGMRKVIEGKGMPIPKYGGYGNLIIKFTIKFPENHFTSEENLKKLEEILPPRIVPA IPKKATVDECVLADFDPAKYNRTRASRGGANYDSDEEEQGGEGVQCASQ*
YDJl is encoded by a non-essential gene comprising an ORF that is 1.230kbp in size and is located on chromosome XTV. A published nucleotide coding sequence of YDJl "is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences whichencode anidenticalproteinproduct:
ATGGTTAAAGAAACTAAGTTTTACGATATTCTAGGTGTTCCAGTAACTGCCACTGATGTC GAAATTAAGAAAGCTTATAGAAAATGCGCCTTAAAATACCATCCAGATAAGAATCCAAGT GAGGAAGCTGCAGAAAAGTTCAAAGAAGCTTCAGCAGCCTATGAAATTTTATCAGATCCT GAAAAGAGAGATATATATGACCAATTTGGTGAAGATGGTCTAΆGTGGTGCTGGTGGCGCT GGCGGATTCCCAGGTGGTGGATTCGGTTTTGGTGACGATATCTTTTCCCAATTCTTTGGT GCTGGTGGCGCACAAAGACCAAGAGGTCCCCAAAGAGGTAAAGATATCAAGCATGAAATT TCTGCCTCACTTGAAGAATTATATAAGGGTAGGACAGCTAAGTTAGCCCTTAACAAACAG ATCCTATGTAAAGAATGTGAAGGTCGTGGTGGTAAGAAAGGCGCCGTCAAGAAGTGTACC AGCTGTAATGGTCAAGGTATTAAATTTGTAACAAGACAAATGGGTCCAATGATCCAAAGA TTCCAAACAGAGTGTGATGTCTGTCACGGTACTGGTGATATCATTGATCCTAAGGATCGT TGTAAATCTTGTAACGGTAAGAAAGTTGAAAACGAAAGGAAGATCCTAGAAGTCCATGTC GAACCAGGTATGAAAGATGGTCAAAGAATCGTTTTCAAAGGTGAAGCTGACCAAGCCCCA GATGTCATTCCAGGTGATGTTGTCTTCATAGTTTCTGAGAGACCACACAAGAGCTTCAAG AGAGATGGTGATGATTTAGTATATGAGGCTGAAATTGATCTATTGACTGCTATCGCTGGT GGTGAATTTGCATTGGAACATGTTTCTGGTGATTGGTTAAAGGTCGGTATTGTTCCAGGT GAAGTTATTGCCCCAGGTATGCGTAAGGTCATCGAAGGTAAAGGTATGCCAATTCCAAΆΆ TACGGTGGCTATGGTAATTTAATCATCAAATTTACTATCAAGTTCCCAGAAAACCATTTC ACATCAGAAGAAAACTTGAAGAAGTTAGAAGAAATTTTGCCTCCAAGAATTGTCCCAGCC ATTCCAAAGAAAGCTACTGTGGACGAATGTGTACTCGCAGACTTTGACCCAGCCAAATAC AACAGAACACGGGCCTCCAGGGGTGGTGCAAACTATGATTCCGATGAAGAAGAΆCAAGGT GGCGAAGGTGTTCAATGTGCATCTCAATGA Further information on YDJl can be obtained from the URL address http://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000005008
It will be appreciated that, by "YDJl", we include fragments or variants thereof having equivalent YDJl -like activity.
XDJl is another S. cerevisiae helper protein of interest for the present invention. It is a putative chaperone, a homolog of E. coli DnaJ, and is closely related to Ydj Ip. A published protein sequence for the protein Xdj Ip is as follows:
MSGSDRGDRLYDVLGVTRDATVQEIKTAYRKLΆLKHHPDKYVDQDSKEVNEIKFKEITAA YEILSDPEKKSHYDLYGDDNGAASSGGANGFGDEDFMNFFNNFFNNGSHDGNNFPGEYDΆ YEEGNSTSSKDIDIDISLTLKDLYMGKKLKFDLKRQVICIKCHGSGWKPKRKI HVTHDVE CESCAGKGSKERLKRFGPGLVASQWWCEKCNGKGKYTKRPKNPKNFCPDCAGLGLLSKK EIITVNVAPGHHFNDVI TVKGMADEEIDKTTCGDLKFHLTEKQENLEQKQIFLKNFDDGA GEDLYTSITISLSEALTGFEKFLTKTFDDRLLTLSVKPGRWRPGDTIKIANEGWPILDN PHGRCGDLYVFVHIEFPPDNWFNEKSELLAIKTNLPSSSSCASHATVNTEDDSNLTNNET ISNFRIIHTDDLPEGIRPFKPEAQDSAHQKARSSYCCIQ*
XDJl is encoded by a non-essential gene comprising an ORF that is 1.38Okbp in size and is located on chromosome XII. A published nucleotide coding sequence of XDJl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identicalproteinproduct:
ATGAGTGGCAGTGATAGAGGAGACCGGTTATACGATGTGTTGGGGGTGACGAGAGATGCG ACCGTGCAAGAGATTAAAACTGCTTACAGAAAGCTTGCCCTGAAACATCATCCGGACAAG TATGTGGATCAAGACTCAΆΆGGAGGTAAΆTGAAΆTCAAATTCAAΆGAGATCACTGCCGCT TACGAGATCTTGAGCGATCCGGAGAAGAAATCACATTACGACTTGTATGGTGATGATAAT GGTGCCGCTAGCAGCGGTGGCGCTAATGGCTTTGGAGATGAAGATTTTATGAACTTCTTT AΆCAATTTCTTCAATAΆTGGAAGTCACGATGGAAATAATTTCCCTGGCGAGTATGATGCG TACGAAGAGGGCAACTCTACAAGCTCTAAGGATATCGATATCGATATATCTCTTACTTTG AAGGATTTGTACATGGGCAAGAAGCTGAAGTTTGATTTAAAGAGACAGGTCATCTGTATA AAGTGCCACGGTTCTGGCTGGAAΆCCAAΆGAGGAAAΆTTCACGTTACACACGATGTGGAΆ TGTGAATCATGCGCTGGAAAGGGTTCAAAGGAACGTCTGAAGAGGTTTGGTCCCGGTTTG GTAGCTTCGCAATGGGTGGTCTGTGAGAAATGTAATGGTAAGGGGAΆGTACACTAAΆΆGA CCCAAGAATCCAAAGAACTTTTGCCCCGATTGCGCAGGCTTGGGGCTCCTGTCAAAGAΆG GAAATCATCACAGTGAACGTGGCTCCGGGACACCACTTTAACGACGTAATTACAGTCAAG GGGATGGCGGACGAGGAAATCGATAAGACCACATGTGGTGATTTAAAGTTCCATCTCACT GAAAAACAAGAAAACTTGGAGCAGAAGCAAATCTTTTTGAAGAACTTTGACGACGGCGCC GGGGAAGATTTGTATACAAGCATTACCATATCGTTAAGCGAGGCCTTGACGGGATTTGAG AAΆTTTTTGACAAAAACCTTCGACGACAGGTTACTAACATTGAGCGTTAAΆCCTGGCAGA GTAGTAAGACCTGGTGACACCATCAAAATCGCCAATGAAGGTTGGCCCATTTTAGATAAC CCTCATGGCCGGTGCGGCGATCTGTATGTTTTCGTTCATATTGAΆTTTCCACCAGATAAC TGGTTCAATGAAAAATCAGAACTACTAGCAATAAAAACGAATCTGCCGTCATCTTCATCT TGTGCCTCACATGCGACTGTAΆATACTGAAGATGACAGCAACCTGACTAACAACGAAACT ATATCAAATTTCCGGATCATTCACACGGACGATCTTCCAGAAGGGATAAGGCCGTTCAAG CCAGAAGCACAGGATTCAGCGCATCAGAAAGCAAGAAGTTCGTACTGCTGTATCCAATGA
Further information onXDJl canbe obtained fromthe URL address http://db.yeastgenome.org/cgi-bin/singlepagefoπnat?sgdid=S000004080
It will be appreciated that, by "XDJl", we include fragments or variants thereof having equivalentXDJl-like activity.
APJl is another S. cerevisiae helper protein of interest for the present invention. It is a putative chaperone of the HSP40 (DnaJ) family; over expression of which interferes with propagation of the [Psi+] prion. A published protein sequence for the protein Apj Ip is as follows:
MQQNTΞLYDSLNVTAAASTSEIKKAYRNAALKYHPDKNNHTEESKRKFQEICQAYEILKD NRLRALYDQYGTTDEVLIQEQQAQAQRQQAGPFSSSSNFDTEAMSFPDLSPGDLFAQFFN SSATPSSNGSKSSFNFSFNNSSTPSFSFVNGSGVNNLYSSSAKYNSNDEDHHLDRGPDIK HNLKCTLKELYMGKTAKLGLNRTRICSVCDGHGGLKKCTCKTCKGQGIQTQTRRMGPLVQ SWSQTCADCGGAGVFVKNKDICQQCQGLGFIKERKILQVTVQPGSCHNQLIVLTGEGDEV ISTKGGGHEKVIPGDWITILRLKDPNFQVINYSNLICKKCKIDFMTSLCGGWYIEGHP SGKLIKLDIIPGEILKPGCFKTVEDMGMPKFINGVRSGFGHLYVKFDVTYPERLEPENAK KIQNILANDKYIKAERSTMETADSDCYCDLEKSYDSVEEHVLSSFEAPNLNNEVIEDDDL GDLINERDSRKRNNRRFDESNINNNNETKRNKYSSPVSGFYDHDINGY*
APJl is encoded by a non-essential gene comprising an ORF that is 1.587kbp in size and is located on chromosome XTV. A published nucleotide coding sequence of APJl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinproduct:
ATGCAACAAAACACGTCTTTATATGACTCTTTGAACGTTACTGCCGCTGCATCCACATCT GAGATTAAGAAAGCTTACAGGAACGCTGCATTAAΆATATCATCCTGATAAAAACAATCAT ACAGAAGAATCCAAGCGAΆAGTTTCAAGAGATATGCCAGGCATACGAAATACTTAAΆGAC AATCGTTTAAGAGCTTTGTATGACCAGTACGGTACCACAGATGAAGTCCTGATTCAAGAG CAGCAGGCGCAGGCGCAΆCGCCAACAAGCCGGGCCGTTCAGTTCATCCTCAAATTTCGAT ACGGAAGCAATGTCATTCCCGGATCTATCTCCAGGTGATCTTTTCGCGCAGTTTTTTAAT AGTTCTGCTACCCCCTCTTCTAATGGCTCCAAAΆGCAGTTTTAATTTTAGCTTCAATAAT AGCTCTACGCCGAGCTTCTCCTTTGTTAATGGCAGTGGCGTGAACAATCTGTACTCCTCG TCAGCAAAATACAACTCCAACGATGAGGACCATCATTTGGATAGAGGCCCTGATATCAAA CATAATCTAΆAGTGCACATTGAAGGAACTCTACATGGGTAΆGACTGCAAAGTTGGGTTTG AATAGGACAAGGATTTGCAGTGTTTGTGATGGGCACGGTGGTCTAAAGAAATGCACTTGT AAAACATGCAΆΆGGGCAΆGGTATTCAAACCCAAACTAGGCGTATGGGACCTCTAGTACAA AGTTGGTCTCAAACTTGTGCAGATTGCGGGGGTGCCGGGGTTTTTGTCAAAAATAAAGAT ATTTGCCAACAGTGCCAAGGTCTTGGCTTCATTAAGGAGAGGAAGATTCTACAAGTCACC GTTCAACCGGGATCGTGTCATAACCAACTTATAGTACTTACGGGCGAAGGTGACGAAGTT ATTAGTACTAAGGGAGGCGGTCACGAAAAGGTAATACCTGGTGACGTCGTTATCACCATT TTACGTTTAAΆAGATCCGAATTTCCAGGTTATCAACTACTCCAATTTGATATGTAAGAAG TGCAAAATCGACTTCATGACCAGTTTATGTGGAGGCGTAGTTTATATTGAAGGGCACCCT AGCGGTAAGTTGATCAAACTTGATATTATACCTGGCGAGATACTGAAGCCTGGTTGTTTC AAGACTGTTGAGGACATGGGGATGCCCAAGTTTATCAACGGTGTTCGGAGCGGTTTCGGT CATCTATATGTCAAA.TTCGATGTGACGTATCCAGAGAGACTGGAACCTGAAAATGCTAAG AAAATACAAAATATTCTGGCTAATGATAAATACATTAAAGCAGAACGTTCCACCATGGAA ACCGCAGATTCAGACTGCTATTGCGATTTGGAGAAGTCATATGACAGTGTGGAAGAGCAT GTGTTAAGTAGCTTTGAGGCCCCTAATTTAAACAATGAAGTTATTGAAGACGACGACCTT GGTGATTTGATTAATGAAAGAGATTCTCGGAAAAGGAACAACCGTCGATTCGACGAAAGT AATATTAATAATAATAATGAAACGAAACGAAATAAATATTCTTCACCGGTAAGCGGTTTT TATGACCATGATATTAΆTGGATATTGA
Further information onAPJl canbe obtained fromtheURL address http://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000005021
It will be appreciated that, by "APJl", we include fragments or variants thereof having equivalentAPJl-like activity. SISl is anotherS. cerevisiae helper protein ofinterest forthe present invention. It is a type II HSP40 co-chaperone that interacts with the HSP70 protein Ssalp; not functionally redundant with Ydjlp due to due to substrate specificity; shares similarity with bacterial DnaJ proteins. A published protein sequence for the protein Sislp is as follows:
MVKETKLYDLLGVSPSANEQELKKGYRKAALKYHPDKPTGDTEKFKEISEAFEILNDPQK REIYDQYGLEAARSGGPSFGPGGPGGAGGAGGFPGGAGGFSGGHAFSNEDAFNIFSQFFG GSSPFGGADDSGFSFSSYPSGGGAGMGGMPGGMGGMHGGMGGMPGGFRSASSSPTYPEEE TVQVNLPVSLEDLFVGKKKSFKIGRKGPHGASEKTQIDIQLKPGWKAGTKITYKNQGDYN PQTGRRKTLQFVIQEKSHPNFKRDGDDLIYTLPLSFKESLLGFSKTIQTIDGRTLPLSRV QPVQPSQTSTYPGQGMPTPKNPSQRGNLIVKYKVDYPISLNDAQKRAIDENF*
SISl is encoded by a non-essential gene comprising an ORF that is 1.059kbp in size and is located on chromosome XIV. A published nucleotide coding sequence of SISl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
ATGGTCAAGGAGACAAAACTTTATGATTTACTTGGAGTATCTCCAAGTGCTAATGAGCAA GAACTGAAΆΆAGGGTTATAGAAAΆGCAGCTCTAAAΆTATCATCCAGATAAGCCAACAGGT GACACAGAAAAGTTTAAGGAGATATCAGAGGCCTTTGAAATTTTAAATGATCCTCAAAAA AGGGAAATATATGATCAATACGGTCTCGAGGCTGCTAGATCTGGTGGTCCAAGCTTTGGT CCTGGTGGTCCTGGCGGTGCTGGAGGTGCTGGAGGCTTCCCTGGCGGTGCGGGCGGATTC TCCGGAGGACATGCGTTCAGTAATGAGGATGCTTTCAATATTTTTTCACAATTCTTTGGC GGCAGTTCCCCATTCGGTGGTGCTGATGACAGTGGCTTCAGTTTCTCTAGTTATCCATCT GGCGGCGGTGCTGGTATGGGAGGTATGCCTGGAGGAATGGGAGGAATGCATGGCGGCATG GGAGGTATGCCTGGCGGCTTTAGATCAGCATCAAGCTCTCCCACGTATCCAGAGGAAGAA ACAGTTCAAGTTAATTTACCAGTTAGTCTAGAAGATTTGTTTGTTGGTAAAAAGAΆGTCA TTTAAAATTGGAAGAAAGGGCCCACΆTGGGGCCTCTGAAAAGACACAΆATTGACATTCAΆ TTAAAACCGGGTTGGAAAGCTGGTACCAAAATAACATACAAGAACCAGGGTGATTACAAT CCTCAAACGGGCCGTAGAAAGACTTTGCAGTTTGTCATCCAGGAAAAGAGCCATCCAΆAC TTTAAAAGAGACGGTGATGACCTAATTTACACTCTGCCACTATCTTTCAAGGAATCATTG TTAGGTTTTTCAAAAACTATCCAAACAATTGATGGCAGAACCTTACCTTTGTCGAGAGTA CAGCCTGTCCAACCCTCACAAACTTCTACTTATCCTGGTCAAGGTATGCCAACTCCAAAG AACCCATCTCAGAGAGGTAATTTGATTGTAAAATATAAAGTGGACTATCCAATATCACTA AACGACGCTCAAΆAACGTGCTATAGATGAAΆATTTTTAA Further information on SISl can be obtained from the URL address http://db.yeastgenome.org/cgi-biri/smglepagefoπΩat?sgdid=S000004952
It will be appreciated that, by "SISl", we include fragments or variants thereof having equivalent SISl -like activity.
DJPl is another S. cerevisiae helper protein of interest for the present invention. It is also known as ICSl and PAS22. It is a J-domain-containing protein, required for peroxisomal protein import and involved in peroxisome assembly, homologous to E. coli DnaJ and is located in the cytoplasm. A published protein sequence for the protein Djplp is as follows:
MVVDTEYYDLLGVSTTASSIEIKKAYRKKSIQEHPDKNPNDPTATERFQAISEAYQVLGD DDLRAKYDKYGRKEAI PQGGFEDAAEQFSVI FGGDAFAS YI GELMLLKNLQKTEELNAE D
EAEKEKENVETMEESPADGKTNGTTNAVDAALGNTNEKDDKNKΆRTTSGNLTVHDGNKKN EQVGAEΆKKKKTKLEQFEEEQEVEKQKRVDQLSKTLIERLSILTESVYDDACKDSFKKKF EEEANLLKMESFGLDILHTIGDVYYEKAEIFLASQNLFGMGGIFHSMKAKGGVFMDTLRT VSAAIDAQNTMKELEKMKEASTNNEPLFDKDGNEQIKPTTEELAQQEQLLMGKVLSAAWH GSKYEITSTLRGVCKKVLEDDSVSKKTLIRRAEAMKLLGEVFKKTFRTKVEQEEAQIFEE LVAEATKKKRHT*
DJPl is encoded by a non-essential gene comprising an ORP that is 1.299kbp in size and is located on chromosome IX. A published nucleotide coding sequence of DJPl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGGTTGTTGATACTGAGTATTACGATTTGTTAGGTGTGTCTACCACTGCATCTTCCATT GAAATAAAAAAGGCCTATAGAAAGAAATCTATTCAAGAGCATCCTGATAAGAATCCCAAT ■
GACCCCACGGCTACCGAAAGGTTTCAAGCAATATCCGAAGCTTATCAAGTTTTAGGTGAC
GATGATCTTCGCGCAAAGTATGATAAGTATGGAAGAAAAGAAGCTATTCCTCAGGGCGGC
TTTGAAGATGCAGCTGAACAGTTCTCTGTCATCTTTGGTGGAGATGCGTTTGCCTCATAT
ATTGGCGAACTGATGCTATTAAAGAACCTACAGAAAACTGAGGAGCTAAATGCTGAAGAC GAAGCTGAAΆAGGAGAΆGGAGAATGTGGAAACAATGGAAGAATCACCTGCAGACGGTAΆG
ACGAATGGCACCACTAACGCTGTTGATGCAGCATTGGGCAATACTAACGAAAAAGATGAC AAAAATAAGGCGAGGACAACTTCTGGTAATTTAACTGTACACGATGGAAACAAGAAAAAT GAGCAGGTAGGAGCAGAAGCTAAGAAGAAGAAGACAAAATTAGAGCAGTTTGAGGAAGAA CAAGAGGTAGAΆAAGCAAAAAAGAGTAGACCAATTAAGCAAAACATTGATTGAAAGATTA TCGATATTAACAGAAAGTGTCTATGATGATGCATGTAAAGATTCCTTTAAAΆAΆAAGTTC GAAGAGGAAGCCAATCTTTTAAAGATGGAATCATTTGGTCTGGACATATTACACACAATA GGCGACGTTTACTACGAAAAAGCTGAAATTTTTCTTGCATCCCAGAACCTGTTCGGAATG GGTGGTATATTTCATTCTATGAAGGCTAAAGGGGGAGTATTTATGGATACACTAAGAACT GTTTCGGCAGCCATAGACGCTCAGAATACTATGAAGGAGCTTGAAAAAΆTGAAAGAAGCT AGCACGAATAATGAGCCTTTGTTTGACAAAGACGGAΆATGAGCAΆATTAAGCCAACCACT GAGGAACTGGCGCAGCAAGAGCAGCTATTGATGGGCAAAGTATTGTCGGCTGCTTGGCAT GGTTCTAAATATGAAATAACATCCACTTTACGTGGCGTTTGTAAAAAAGTACTAGAAGAT GACTCGGTAAGTAAGAΆAACGCTTATCAGAAGAGCTGAAGCAATGAAΆCTATTGGGTGAA GTCTTTAAGAΆAACTTTCAGAACCAAΆGTCGAACAΆGAAGAGGCACAGATCTTTGAAGAΆ CTTGTAGCAGAAGCTACAAAΆAAGAAGAGACATACATGA
Further information on DJPl can be obtained from the URL address http://db.veastgenome.org/cgi-bin/smglepageformat?sgdid:=:S000001443
It will be appreciated that, by "DJPl", we include fragments or variants thereof having equivalent DJP 1 -like activity.
ZUOl is another S. cerevisiae helper protein of interest for the present invention. It is a cytosolic ribosome-associated chaperone that acts, together with Sszlp and the Ssb proteins, as a chaperone for nascent polypeptide chains; contains a DnaJ domain and functions as a J-protein partner for Ssb Ip and Ssb2p. A published protein sequence for the protein Zuolp is as follows:
MFSLPTLTSDITVEVNSSATKTPFVRRPVEPVGKFFLQHAQRTLRNHTWSEFERIEAEKN VKTVDESNVDPDELLFDTELADEDLLTHDARDWKTADLYAAMGLSKLRFRATESQIIKAH RKQWKYHPDKQSAAGGSLDQDGFFKIIQKAFETLTDSNKRAQYDSCDFVADVPPPKKGT DYDFYEAWGPVFEAEARFSKKTPIPSLGNKDSSKKEVEQFYAFWHRFDSWRTFEFLDEDV PDDSSNRDHKRYIERKNKAARDKKKTADNARLVKLVERAVSEDPRIKMFKEEEKKEKERR KWEREAGARAEAEAKAKAEAEAKΆKAESEAKANASAKADKKKAKEAAKAΆKKKNKRAIRN SAKEADYFGDADKATTIDEQVGLIVDSLNDEELVSTADKIKANAAGAKEVLKESAKTIVD SGKLPSSLLSYFV* ZUOl is encoded by a non-essential gene comprising an ORF that is 1.302kbp in size and is located on chromosome VII. A published nucleotide coding sequence of ZUOl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
ΆTGTTTTCTTTACCTACCCTAACCTCAGACATCACTGTTGAAGTCAACAGTTCCGCTACC AAAΆCCCCATTCGTCCGTCGTCCGGTCGAACCGGTTGGTAAGTTCTTTTTGCAACATGCT CAAAGAACTTTGAGAAΆCCACACCTGGTCTGAΆTTTGAAAGAATTGAAGCTGAAAΆGAAC GTCAAAACCGTTGATGAATCCAATGTCGACCCAGATGAGTTGTTATTCGACACTGAATTG GCCGATGAAGATTTACTGACTCATGATGCTAGAGACTGGAAAACTGCCGATTTGTATGCT GCTATGGGTTTGTCTAAGTTGCGTTTCAGAGCTACTGAAAGTCAAATCATCAAGGCTCAC AGAAAACAAGTTGTCAAGTACCATCCAGACAAGCAATCTGCTGCTGGTGGTAGTTTGGAC CAAGATGGCTTTTTCAAGATTATTCAAAAGGCCTTTGAAACTTTGACTGATTCCAACAAG AGAGCTCAGTACGACTCATGTGATTTTGTTGCCGATGTTCCTCCTCCAAAGAAGGGTACC GATTATGACTTTTATGAAGCTTGGGGCCCCGTTTTCGAAGCTGAAGCTCGTTTTTCTAAG AAGACTCCTATTCCTTCTCTAGGTAACAΆAGATTCTTCCAΆGAAGGAAGTTGAACAATTC TATGCTTTCTGGCACAGATTTGACTCCTGGAGAACCTTTGAGTTCTTGGACGAAGATGTC CCAGATGACTCTTCTAACAGAGACCACAΆGCGTTACATTGAAΆGAAΆGAACAAGGCCGCA AGAGACAAGAAGAAGACTGCTGATAACGCTAGATTGGTCAAACTTGTTGAAAGAGCTGTC AGTGAAGATCCCCGTATCAΆAATGTTCAAAGAAGAAGAGAAGAAGGAΆAΆGGAAAGAAGA AAATGGGAAAGAGAAGCCGGTGCCAGAGCTGAAGCTGAAGCTAAGGCCAAGGCCGAAGCT GAAGCGAAGGCTAAAGCTGAATCTGAAGCCAAGGCTAACGCCTCCGCAAAAGCTGACAAA AAGAAGGCTAAGGAAGCTGCTAAGGCCGCCAAGAAAAAGAΆCAΆGAGAGCCATCCGTAAC TCTGCTAAGGAAGCTGACTACTTTGGTGATGCTGACAAGGCCACCACGATTGACGAACAA GTTGGTTTGATCGTTGACAGTTTGAATGACGAAGAGTTAGTGTCCACCGCCGATAAGATC AAGGCCAATGCTGCTGGTGCCAAGGAAGTTTTGAAGGAATCTGCAAAGACTATTGTCGAT TCTGGCAAACTACCATCCAGCTTGTTGTCCTACTTCGTGTGA
Furtherinformation on ZUOl canbe obtained fromtheURL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid==:S000003517
It will be appreciated that, by "ZUOl", we include fragments or variants thereof having equivalentZUO1-like activity.
SWA2 is another S. cerevisiae helper protein ofinterest for the present invention.
It is also known as AUXl and BUD24. It is an auxilin-like protein involved in vesicular transport; clathrin-binding protein required for uncoating of clathrin- coated vesicles. Apublishedprotein sequence forthe protein Swa2p is as follows:
MSDPFAHLLTSLKNKDSASASKETTPQSSNSPSITGSAVADVARTDKSPNDSLHSISAPP LIPSPKVDFSAPPLVPTNSTTKSNTANNTPPSALANTDDDFNQLFGMGTVTTTDTIQKPD EDYYGSKEDHLYNGDDALVDEVKDMEIARLMSLGLSIEEATEFYENDVTYERYLEILKSK QKERNDLΆIRKKESGIKMEKSGLSNIVGTDSNNLFSMATDFFNKGKKLVDQWTSFPPEAN DRLNNYSKTHDKVEDYDLPQVNDSPNRILFEDNEVVENLPPADNPDQDLLTDFETKIDIT KRTAPDVSHSSSPTSGILIEENSRRNEPLIEDSLLDFSEGNLTNSKSNEDSTLFNENSNT DSTIPISDIELSGYNEFKAKGTSLFKNGDYINSLQEYEKSLNTLPLNHPLRIIALSNIIA SQLKIGEYSKSIENSSMALELFPSSKAKWKNKISNSDPERSFNDIWPKIMIRRAESFEHL ESFKKALETYQELIKKNFFDDKIMQGKRRCQDFINPPPVKKSMPVKKKTTTTSPATKKQN LTASSSNSPISVDSTSEIKKRELENAKLALYDKVFEKISSWKDGKDDDIRHLLANLSSLL TWCNWKDVSMQDLVMPKRVKITYMKAVAKTHPDKIPESLSLENKMIAENIFSTLSIAWDK FKLQNDIN*
SWA2 is encoded by a non-essential gene comprising an ORP that is 2.007kbp in size and is located on chromosome IV. A published nucleotide coding sequence of SWA2 is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidenticalproteinproduct:
ATGTCAGATCCATTTGCACATTTACTGACTTCTTTGAAGAATAAGGACTCTGCATCTGCA TCCAAGGAAACAACTCCTCAGAGCAGCAATTCGCCTTCCATTACTGGTTCCGCTGTTGCA GATGTTGCAAGGACGGATAAAAGCCCCAATGATAGTCTGCATTCAATTTCAGCTCCTCCG . CTGATACCGTCACCGAAGGTAGATTTTTCTGCACCTCCTTTGGTCCCAACTAATAGCACC ACTAAATCTAΆTACTGCCAACAACACACCTCCCTCGGCTCTTGCCAATACCGATGACGAC TTCAΆTCAACTATTTGGTATGGGCACAGTAACTACAACGGATACGATCCAAAAΆCCGGAT GAGGATTACTATGGAAGCAAGGAAGACCACCTTTACAATGGTGATGACGCCTTAGTTGAT GAAGTTAAGGATATGGAAATAGCAAGATTGATGTCTCTAGGTTTATCAATTGAAGAAGCC ACTGAGTTTTACGAAAΆTGACGTAACTTATGAAAGATATTTGGAGATTTTAAAGTCAAAG CAAAAGGAGCGCAΆCGATCTAGCTATAAGAAΆGAAAGAAAGTGGTATAAΆAATGGAAΆΆG TCAGGATTATCCAACATTGTTGGTACAGATAGCAATAATTTATTCAGCATGGCCACTGAT TTTTTCAATAAGGGTAAGAAACTGGTAGACCAATGGACCTCCTTCCCACCTGAGGCAAAT GATAGACTGAATAATTACTCAAΆAΆCTCATGATAAGGTTGAGGATTATGATTTGCCTCAΆ GTAΆACGACTCACCCAATAGAATTTTGTTTGAΆGATAΆTGAAGTCGTAGAGAACTTACCA CCTGCCGATAATCCGGATCAAGATCTTTTAACTGATTTCGAAACAAAGATTGATATAΆCA AAGAGGACAGCGCCTGATGTCTCCCACTCCTCCTCACCGACTTCTGGTATACTAATTGAA GAAAATTCGCGAAGAT-ATGAGCCCCTGATAGAGGATAGTCTTCTCGACTTTTCAGAAGGA
AATCTCACCAΆTAGTAAAAGCAATGAAGATAGCACCCTCTTCAATGAAAΆCAGCAACACT GACTCTACAATACCCATCTCAGΆTATTGAATTATCGGGGTATAACGAATTTAAGGCGAAΆ GGTACTAGTTTGTTCAAGAACGGGGATTATATTAACTCATTACAAGAATATGAAAAGTCT TTAAATACATTGCCTTTAAATCATCCATTGAGGATCATTGCATTATCAAACATTATTGCC TCGCAACTGAAAATCGGTGAGTACTCTAAGTCCATAGAAΆΆCTCCAGCATGGCTTTGGAΆ TTATTCCCATCAAGCAΆAGCTAAGTGGAAGAATAAAATCTCAAΆTAGTGACCCTGAAAGA TCATTTAACGACATCTGGCCAAAGATTATGATTAGGCGTGCTGAGTCTTTTGAACATTTA GAAAGTTTCAAAAAAGCACTAGAAACATACCAAGAGCTGATTAAGAAGAATTTTTTTGAT GATAAAATCATGCAGGGAAAAAGAAGATGCCAAGACTTTATTAATCCTCCCCCTGTTAAA AAATCCATGCCCGTTAAGAAGAAGACAΆCGACAACCTCGCCTGCAΆCAAΆΆAAACAGAAC TTAACCGCTTCTTCTTCAΆATTCTCCAΆTTTCTGTTGATAGCACTTCAGAAATAAAAAAA CGGGAGCTAGAAAACGCTAAACTGGCGCTATATGATAAΆGTATTTGAGAAAATTAGCTCC TGGAAGGATGGCAAAGACGATGACATTCGTCATCTGTTAGCAAATTTATCCAGCTTACTA ACATGGTGCAATTGGAAGGATGTCTCTATGCAAGATTTGGTTATGCCTAAGAGGGTCAAA ATTACATACATGAAAGCTGTAGCCAAGACACATCCTGATAAGATACCAGΆGTCCTTGTCC CTGGAAAATAAGATGATTGCAGAGAATATTTTCAGTACTTTAAGTATTGCTTGGGATAAG TTCAAΆCTGCAGAATGACATTAACTGA
Further information on SWA2 canbe obtained fromthe URL address http://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000002728
It will be appreciated that, by "SWA2", we include fragments or variants thereof having equivalent SWA2-like activity.
JJJl is another S. cerevisiae helper protein of interest for the present invention. It contains a 70 amino acid J-domain, may function as a co-chaperone to recruit Hsp70-like activity to specific sites; mutation of it causes defects in fluid-phase endocytosis. A published protein sequence for the protein Jjj Ip is as follows:
MKTCYYELLGVETHASDLELKKAYRKKALQYHPDKNPDNVEEATQKFAVIRAAYEVLSDP QERAWYDSHKEQILNDTPPSTDDYYDYEVDATVTGVTTDELLLFFNSALYTKIDNSAAGI YQIAGKIFAKLAKDEILSGKRLGKFSEYQDDVFEQDINSIGYLKACDNFINKTDKLLYPL FGYSPTDYEYLKHFYKTWSAFNTLKSFSWKDEYMYSKNYDRRTKREVNRRNEKARQQARN EYNKTVKRFWFIKKLDKRMKEGAKIAEEQRKLKEQQRKNELNNRRKFGNDNNDEEKFHL QSWQTVKEENWDELEKVYDNFGEFENSKNDKEGEVLIYECFICNKTFKSEKQLKNHINTK LHKKNMEEIRKEMEEENITLGLDNLSDLEKFDSADESVKEKEDIDLQALQAELAEIERKL AESSSEDESEDDNLNIEMDIEVEDVSSDENVHVNTKNKKKRKKKKKAKVDTETEESESFD DTKDKRSNELDDLLASLGDKGLQTDDDEDWSTKAKKKKGKQPKKNSKSTKSTPSLSTLPS SMSPTSAIEVCTTCGESFDSRNKLFNHVKIAGHAAVKNVVKRKKVKTKRI*
JJJl is encoded by a non-essential gene comprising an ORF that is 1.773kbp in size and is located on chromosome XIV. A published nucleotide coding sequence of JJJl is as follows, 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:
ATGAAGACCTGCTACTATGAGCTTTTAGGGGTCGAAACGCATGCTTCTGATCTTGAGTTA AAAAAAGCTTACCGTAΆAΆAGGCCCTACAATATCACCCAGATAAAΆACCCAGATAATGTT GAΆGAAGCCACACAAAAATTTGCTGTGATTCGAGCCGCTTATGAΆGTACTGTCTGACCCC CAGGAAAGAGCATGGTATGACTCACATAAGGAΆCAΆATTTTAAATGATACTCCACCAAGC ACTGATGATTACTATGATTATGAGGTAGACGCTACAGTCACAGGTGTCACAACTGATGAA TTACTCTTATTTTTTAACTCTGCTCTTTATACTAAAATAGACAACTCAGCTGCTGGGATΆ TATCAΆATTGCAGGAAΆAATATTTGCCAAGTTAGCTAAAGATGAGATTTTAAGTGGTAAG CGACTGGGGAAΆTTTTCCGAGTATCAAGATGATGTATTCGAACAGGATATTAΆTAGTATT GGCTATTTGAAAGCCTGCGATAACTTTATTAACAAGACGGATAAACTTTTATATCCTTTA TTTGGATATTCGCCAACGGATTATGAATATTTGAAACATTTCTATAAGACTTGGTCAGCG TTCAATACCTTGAAAAGTTTTAGCTGGAAAGACGAGTACATGTACTCTAAAAACTATGAC AGAΆGAACCAAGAGGGAAGTTAATAGAAGAAΆTGAGAAGGCTAGGCAACAAGCTCGAAAT GAATACAACAAAACCGTGAAAΆGGTTTGTAGTTTTCATAAAAAAGCTCGATAAAΆGAATG AΆAGAAGGTGCAΆAAATTGCAGAAGAACAGCGTAAACTAΆAAGAACAACAGAGGAAΆAΆT GAGTTAAATAACAGAAGAAAGTTTGGGAACGACAACAATGACGAAGAAAAATTTCATTTA CAAAGCTGGCAAACGGTAAAAGAAGAAAACTGGGATGAACTGGAΆAΆGGTATATGATAAT TTTGGAGAATTCGAAAATTCTAAGAATGATAAGGAAGGTGAAGTATTGATTTACGAGTGT TTTATCTGCAACAΆGACATTTAAGTCGGAAAAGCAATTGAΆΆAACCACATAAΆCACTAAΆ CTGCATAAGAAAAATATGGAAGAGATACGGAAAGAAATGGAAGAGGAAAACATAACGCTT GGGTTGGATAATCTCTCCGATCTCGAGAAATTTGATTCAGCAGATGAAAGTGTTAAAGAA AAAGAAGATATTGATCTGCAAGCATTGCΆAGCTGAACTCGCTGAAATTGAAAGAAAACTG GCAGAATCGTCTTCTGAAGACGAAAGTGAAGATGACAATCTCAACATAGAAATGGATATA GAGGTAGAAGACGTCAGTTCGGATGAΆAATGTACATGTGAΆTACGAΆGAATAAAAΆGAAA AGAAAAAAGAAAΆAAAAAGCAAAGGTTGΆCACAGAAACAGAGGAATCTGAATCGTTCGAT GATACTAAΆGACAAΆCGGAGTAΆTGAGTTGGATGATCTTTTGGCATCACTAGGAGACAAG GGCTTACAAACGGATGACGATGAΆGATTGGTCTACTAAAGCGAAAAAGAAAAΆGGGCAAA CAACCTAAAΆΆGAATTCTAAATCCACAAAAAGCACTCCGTCCTTGTCGACTCTACCGTCC TCTATGTCTCCAACCTCCGCGATCGAGGTGTGCACTACATGCGGAGAATCATTTGATAGT CGAAATAAGCTATTCAACCACGTGAAGATAGCAGGGCATGCGGCAGTGAAAAACGTAGTG AAAAGAAAGAAΆGTCAAGACCAAAAGAATATAG
Further information on JJJl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid=rS000005171
It will be appreciated that, by "JJJl", we include fragments or variants thereof having equivalent JJJl -like activity.
JJJ2 is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the cytoplasm. A published protein sequence for the protein Jjj2p is as follows:
MSQVIEPQLDRTTYYSILGLTSNATSSEVHKSYLKLARLLHPDKTKSDKSEELFKAVVHA HSILTDEDQKLRYDRDLKIKGLHTYQPKKNCHIFKTKAKESQGASPTLGQSEAYHRQNKP
YEQQPYGFGVGKKMTSSSKSKVPIFKSFNLKSYQRNHYYSSKKERKHGSPDIDSLFHETN
GASKVRMTDAGKMDTNSQFQEIWEILGKNAYTHKSYSEDPNSCLGSALSDHEEEEEΆGKQ
QQQQQQQQQQQQHYGMTSKSSSPDEEKKNNKEPKRESRVSPEENGEEETGHKQFKLPKTS
TFSSGSHDSNLQSPFYNHEYRHYARSKFECKNQFRKSVSPIKEIPATTSANEGWNILRDI IEKLNISNVDDRNKDLLFRRDEIGDKNHSDSIDIENLSIKEPKGMKRRKKDDISLEELFQ SLPREKDYFMMDAINDSLESINLFKKPKTTQSHEQGGTFAQAESNRAKFKPLLEQCGITP
EILDLEIPEIPEFDAVADLETLKLNVQLFNNQCNKLKETIHQVSLQRLRADTQFSDMLTQ
KQSIMVWKTYLEFDKSLMDKLNILQERQMQVIKIFSERCDGKV*
JJJ2 is encoded by a non-essential gene comprising an ORP that is 1.752kbp in size and is located on chromosome 10. A published nucleotide coding sequence of JJJ2 is as follows, 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:
ATGTCACAGGTAATAGAACCACAATTAGATAGAACAACCTATTATTCCATATTAGGCTTG ACATCAAATGCGACTTCCTCCGAAGTACATAAATCATATCTAAAACTGGCCAGATTACTT CACCCAGATAAΆACAAAATCTGATAAGTCTGΆGGAATTATTCAAAGCTGTGGTGCATGCA CATTCAΆTTTTAACTGATGAAGATCAAAAACTTCGATATGATCGAGATTTGAΆAATCAAA GGTTTACACACTTACCAGCCGAAGAAAAACTGTCATATTTTCAAGACCAAGGCAAAGGAA TCACAΆGGGGCTAGTCCCACACTTGGTCAATCAGAAGCTTATCATAGGCAAAATAΆACCT TATGAGCAACAGCCCTACGGTTTCGGTGTAGGCAAAAAAATGACCTCAAGCTCTAAGAGT AAGGTTCCGATATTCAAGTCCTTCAΆTTTAAAAΆGCTACCAACGAAACCACTATTATTCA TCCAAΆAΆGGAAΆGGAΆΆCATGGAAGTCCTGATATTGATTCTTTGTTCCATGAAΆCCAAT GGAGCCTCAAAΆGTAAGAATGACTGATGCCGGTAAΆATGGATACGΆACTCTCAGTTCCAA GAAATATGGGAAATATTGGGTAAAAATGCGTACACACATAAATCTTACTCTGAAGATCCA AATTCATGTTTGGGATCAGCACTAAGCGATCATGAAGAAGAAGAAGAAGCAGGAAAACAA CAACAGCAACAGCAGCAACAACAGCAACAGCAGCAACATTATGGAATGACGTCGAΆGTCT AGCAGTCCTGATGAΆGAAAAΆΆAAAATAATAAAGAΆCCGAAΆAGGGAAAGCAGAGTCTCT CCAGAGGAAAATGGCGAAGΆAGAAACGGGACACAAACAATTTAAATTGCCCAΆGACCAGT ACTTTTTCTAGTGGATCCCATGATTCAAATTTGCAATCTCCTTTTTACAATCATGAGTAT CGACATTACGCAAGAAGTAAATTCGAATGCAAGAATCAGTTTAGAAAGTCAGTTTCTCCC ATTAΆAGAGATACCTGCAACAACTAGTGCCAATGAAGGATGGAACATTTTGAGAGACATT ATTGAAAAACTCAATATAAGCAATGTAGACGATCGAAATAΆAGACTTGCTGTTTCGTCGG GATGAAΆTAGGTGATAAAAATCACAGCGACTCAATCGACATAGAAAATTTATCTATCAAA GAACCTAAΆGGGATGAAAAGGAGAΆAGAAAGATGATATATCTTTAGAAGAΆTTGTTCCAA TCTTTACCAAGAGAAAAΆGATTATTTTATGATGGATGCAATTAATGACTCGTTAGAATCA ATCAATCTTTTTAAAAAGCCGAAGACCACTCAGAGTCACGAACAAGGTGGAACTTTTGCC CAAGCAGAAAGTAATCGTGCAAAΆTTCAAACCGTTACTAGAΆCAGTGTGGAATTACACCC GAGATCTTAGATTTGGAAATACCAGAGATTCCGGAATTTGATGCAGTGGCTGACCTTGAA ACATTGAAGCTTAACGTGCAGCTGTTTAATAACCAATGTAACAAACTTAAAGAAACAATA CATCAAGTATCATTACAGCGCCTGAGAGCAGATACGCAGTTCAGTGATATGTTAACCCΆA AAGCAAAGTATTATGGTTTGGAAAΆCATACCTAGAATTTGATAAAAGTTTAATGGACAAA TTGAACATCTTACAAGAAAGACAGATGCAGGTCATTAAAΆTTTTTTCCGAAΆGATGTGAC GGTAAAGTATAA
Further information on JJJ2 can be obtained from the URL address http://db.yeastgenome.Org/cgi-bin/singlepao:eformat7sgdid~S000003698
It will be appreciated that, by "JJJ2", we include fragments or variants thereof having equivalent JJJ2-like activity.
JJJ3 is another S. cerevisiae helper protein of interest for the present invention and is also known as DPH4. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the cytoplasm. A published protein sequence for the protein Jjj3p is as follows:
MSLVNSLTHYEILRIPSDATQDEIKKAYRNRLLNTHPDKLSKSIHDTVSNVTINKIQDAY KILSNIKTRREYDRLILENYKRQGFHNCGDGLDEFSLDDFSFDEDKLEFMMNCPRCQFVG GFHFSESLLDECIDNVDAMERSHSGYQLLTQCSACSLWLKVNFDIEEEQEGQ
JJ.T3 is encoded by a non-essential gene comprising an ORF that is 0.519kbp in size and is located on chromosome X. A published nucleotide coding sequence of JJJ3 is as follows, 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:
ATGTCATTGGTGAATTCGTTAACACACTACGAAATTTTAAGAATTCCATCGGATGCAACA CAAGATGAAATCAAAAΆGGCATATAGGAATCGGTTACTAAATACGCACCCCGATAAACTT TCTAAAΆGCATACATGATACGGTTAGCAΆCGTCACAATCAATAΆGATTCAAGATGCTTAT AAAATACTATCGAATATAAAAACTCGTCGCGAATATGATAGGTTGATCCTTGAAAACTAT AAΆCGCCAΆGGATTTCATAATTGTGGTGATGGGCTGGATGAATTTTCCTTAGACGATTTC TCATTTGATGAAGATAAGCTGGAGTTTATGATGAATTGTCCTCGCTGTCAATTTGTTGGT GGTTTTCATTTTAGTGAGAGTTTGTTAGATGAATGCATTGATAATGTAGACGCTATGGAA CGGAGTCATTCTGGTTATCAATTATTAACCCAATGTAGCGCATGCAGCTTATGGCTGAAG GTTAATTTTGACATCGAGGAΆGAGCAΆGAAGGACAATAA
Further information on JJJ3 can be obtained from the URL address b.ttp://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000003858
It will be appreciated that, by "JJJ3", we include fragments or variants thereof having equivalent JJ J3 -like activity.
CAJl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the nucleus. A published protein sequence for the protein Caj Ip is as follows:
MVKETEYYDILGIKPEATPTEIKKAYRRKAMETHPDKHPDDPDAQAKFQAVGEAYQVLSD PGLRSKYDQFGKEDAVPQQGFEDASEYFTAIFGGDGFKDWIGEFSLFKELNEATEMFGKE DEEGTAATETEKADESTDGGMVKHDTNKAESLKKDKLSKEQREKLMEMEKKRREDMMKQV DELAEKLNEKISRYLIAVKSNNLEEFTRKLDQEIEDLKLESFGLELLYLLARVYKTKANN
FIMSKKTYGISKIFTGTRDNARSVKSAYNLLSTGLEAQKAMEKMSEVNTDELDQYERAKF ESTMAGKALGVMWAMSKFELERKLKDVCNKILNDKKVPSKERIAKAKAMLFIAHKFASAR RSPEEAEEARVFEELILGEQEKEHKKHTVAR CAJl is encoded by a non-essential gene comprising an OKF that is 1.176 kbp in size and is located on chromosome V. A published nucleotide coding sequence of CAJl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode an identical proteinprodμct:
ATGGTAAAGGAGACGGAGTATTATGATATTTTGGGCATCAAGCCTGAGGCCACGCCCACT GAAATCAAAAAGGCCTATCGTAGAAAGGCTATGGAAACACATCCGGACAAGCATCCTGAT GACCCAGATGCTCAAGCAAAGTTTCAAGCCGTAGGCGAGGCCTACCAAGTCTTAAGTGAT CCAGGGCTTCGTTCCAAGTATGACCAGTTTGGTAAGGAGGATGCTGTTCCTCAGCAAGGA TTTGAAGATGCTTCTGAATACTTTACAGCAATATTCGGTGGTGATGGCTTCAΆAGATTGG ATTGGAGAATTTTCTTTGTTCAΆAGAGCTAAACGAGGCAACAGAΆATGTTTGGAAΆGGAA GATGAGGAGGGTACAGCAGCCACTGAAACCGAAAAAGCAGATGAGAGCACTGATGGTGGA ATGGTTAAGCATGACACTAATAAAGCTGAATCTTTGAAAAAAGATAAATTATCGAAGGAG CAAΆGAGAGAAGCTAATGGAAATGGAGAAAAAAAGACGGGAAGATATGATGAΆACAAGTC GACGAGTTGGCAGAAAAACTGAACGAAAAAATCTCTAGGTACTTAATTGCTGTGAAGTCC AATAACTTGGAGGAATTTACGCGAAAACTAGATCAAGAAATCGAGGATTTGAAATTAGAA AGTTTTGGTCTAGAGTTATTGTATTTATTGGCCAGGGTTTACAAGACAAAAGCGAATAAT TTTATCATGTCCAAGAAGACTTACGGAATTTCTAAAATATTCACTGGTACACGCGACAAT GCTAGATCTGTTAAATCAGCATACAATTTATTGTCTACAGGCTTAGAΆGCTCAAAΆAGCC ATGGAAAAAATGAGTGAAGTCAATACTGACGAACTAGACCAATATGAACGTGCCAAATTT GAGTCCACAATGGCTGGTAAGGCACTTGGTGTCATGTGGGCTATGTCGAAATTTGAACTG GAAAGAAAACTAAAAGACGTTTGCAATAAGATTCTAAACGATAΆAΆΆGGTCCCTTCCAAG GAACGTATTGCAAAGGCAAAAGCAATGCTGTTTATTGCCCACAAGTTTGCCAGTGCTAGA AGGTCACCAGAAGAAGCTGAAGAAGCTAGAGTTTTTGAAGAGCTAATCCTAGGTGAGCAG GAGAΆGGAACACAAAAAΆCATACTGTGGCCAGATAA
Further information on CAJl canbe obtained fromthe URL address http://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000000850
It will be appreciated that, by "CAJl", we include fragments or variants thereof having equivalent CAJl-like activity.
CWC23 is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs ofthe bacterial chaperone DnaJ, and is located in the nucleus. A published protein sequence for the protein Cwc23p is as follows:
MPGHELEDVINQRLNLYDVLELPTPLDVHTIYDDLPQIKRKYRTLALKYHPDKHPDNPSI IHKFHLLSTATNILTNADVRPHYDRWLIEFLRKTNDIERNKLIQKLEESESSTIPTTTPH PDLLQIQRHGELLRKLKHFNLPYGDWKHLNTQDQENASQHPYYDCSTLRIVLDNFLQSNN KSNCLSHLRNQVFITLSANEIYDIYFSERNNYSKDDSIIIYTVFDTPITAQHVFRNWSSG NLIPTVKDISPLIPLHYYSDFNLETELNDDIARLVSNEPILLD
CWC23 is encoded by an essential gene comprising an ORP that is 0.852 kbp in size and is located on chromosome VII. A published nucleotide coding sequence of CWC23 is as follows, 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:
ATGCCAGGACACGAATTGGAAGACGTAATAAATCAACGTTTGAACCTATATGATGTATTA GAATTACCGACCCCCCTGGACGTCCATACCATCTACGATGATTTGCCCCAAATTAAACGC AAATACAGGACCCTTGCCCTGAAGTATCATCCTGACAAACACCCGGACAATCCATCAATT ATACACAAATTCCACTTATTATCGACCGCAACTAATATCCTCACCAATGCAGACGTGΆGA CCCCATTACGACCGCTGGTTAATTGAGTTCCTACGGAAAACAAACGACATTGAAAGAAAT AAACTTATACAAAAGCTGGAAGAATCTGAATCGAGTACGATACCCACCACCACACCACAT CCTGATTTATTGCAAATCCAACGCCACGGCGAGCTACTCAGGAAACTAAAACATTTCAAC TTGCCCTATGGTGACTGGAAACATCTCAACACACAAGACCAAGAAΆATGCTTCGCAΆCAT CCGTATTACGATTGCTCTACTTTGAGAATTGTCCTTGACAACTTCCTGCAATCAAATAAT AAATCAAACTGCTTATCTCATTTGCGCAATCAAGTATTCATCACGCTAAGTGCTAATGAA ATCTACGACATCTACTTCTCTGAΆAGAAACAACTACTCGAΆGGATGATTCAATCATCATA TATACTGTATTCGATACTCCCATCACAGCGCAGCACGTATTCCGAAACTGGTCAAGTGGG AACCTCATACCCACGGTCAAGGATATTTCGCCCTTGATCCCGCTACATTACTACTCTGAT TTTAATTTGGAGACGGAACTGAATGACGATATTGCAAGACTGGTCTCTAATGAACCTATC CTACTCGACTAG
Further information on CWC23 can be obtained from the URL address http://db.veastgenome.org/cgi-bin/smglepageformat?sgdid=S000003096
It will be appreciated that, by "CWC23", we include fragments or variants thereof having equivalent CWC23-like activity. PAM18 is another 5*. cerevisiae helper protein of interest for the present invention and is also known as TIMl 4. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the mitochondria. A published protein sequence for the protein Paml 8p is as follows:
MSSQSNTGNSIEAPQLPIPGQTNGSANVTVDGAGVNVGIQNGSQGQKTGMDLYFDQALNY MGEHPVITGFGAFLTLYFTAGAYKSISKGLNGGKSTTAFLKGGFDPKMNSKEALQILNLT ENTLTKKKLKEVHRKIMLANHPDKGGSPFLATKINEAKDFLEKRGISK
PAMl 8 is encoded by an essential gene comprising an ORP that is 0.507 kbp in size and is located on chromosome XII. A published nucleotide coding sequence of PAMl 8 is as follows, 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:
ATGAGTTCTCAAAGTAATACTGGTAATTCTATTGAGGCACCACAACTACCCATTCCTGGT CAAACTAATGGCTCTGCGAACGTTACTGTTGATGGAGCTGGTGTTAATGTCGGTATCCAG AATGGTTCGCAGGGTCAAAΆGACCGGAATGGACCTTTATTTTGATCAAGCTTTGAACTAC ATGGGAGAACATCCTGTGATAACAGGTTTTGGGGCCTTTTTAACTTTATATTTTACAGCC GGTGCATATAAATCAATATCGAΆGGGACTTAACGGTGGAAΆATCCACTACTGCCTTCTTG AAΆGGCGGATTTGACCCGAAAATGAATTCTAAAGAGGCTCTACAGATTTTGAATTTGACA GAAAATACATTGACTAAAAAAAAGTTGAAAGAGGTTCATAGGAAAATTATGTTAGCTAAT CATCCTGACAΆAGGTGGTTCTCCATTTTTGGCCACTAAGATAAACGAAGCTAAGGACTTT TTGGAAAAAAGGGGTATTAGCAAATAA
Further information on PAMl 8 can be obtained from the URL address http://db.veastgenome.org/cgi-bin/singlepageformat?sgdid=S000003998
It will be appreciated that, by "PAMl 8", we include fragments or variants thereof having equivalent PAM18-like activity. JACl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the mitochondria. A published protein sequence for the protein Jaclp is as follows:
MLKYLVQRRFTSTFYELFPKTFPKKLPIWTIDQSRLRKEYRQLQAQHHPDMAQQGSEQSS TLNQAYHTLKDPLRRSQYMLKLLRNIDLTQEQTSNEVTTSDPQLLLKVLDIHDELSQMDD EAGVKLLEKQNKERIQDIEAQLGQCYNDKDYAAAVKLTVELKYWYNLAKAFKDWAPGKQL EMNH
JACl is encoded by an essential gene comprising an ORF that is 0.555 kbp in size and is located on chromosome VII. A published nucleotide coding sequence of JACl is as follows, 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:
ATGTTGAAATACTTGGTTCAACGAAGATTCACTTCTACATTTTACGAGCTGTTCCCAAAG ACCTTCCCCAAAAAGCTACCCATTTGGACTATCGATCAATCCAGATTAAGGAAGGAGTAT AGGCAATTACAAGCACAGCACCATCCAGACATGGCCCAACAAGGTAGTGAACAGTCATCA ACTCTTAATCAAGCTTACCATACTCTCAAΆGATCCCCTTAGAΆGGTCACAATATATGCTA AAACTCTTGCGCAATATCGATTTGACGCAΆGAACAGACCTCAAATGAAGTAΆCTACCAGT GATCCACAGTTACTATTGAAAGTTCTAGACATCCATGATGAATTATCCCAGATGGACGAC GAAGCTGGTGTGAAGCTGCTTGAAAAGCAAAACAAGGAAAGAATTCAAGATATTGAAGCC CAGTTGGGACAΆTGCTACAATGACAAGGATTACGCCGCCGCAGTGAAGTTGACCGTGGAG CTAAΆGTACTGGTACAACTTGGCCAAGGCATTCAAΆGACTGGGCTCCAGGAAAACAΆTTG GAAATGAATCACTAA
Furtherinformationon JACl canbe obtained fromtheURL address http://dh.veastgenome.org/cgi-bin/singlepageformat?sgdid:=S000002986
It will be appreciated that, by "JACl", we include fragments or variants thereof having equivalentJACl-like activity. • JIDl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the mitochondria. A published protein sequence for the protein Jidlp is as follows:
MLHHKFVYPFLFKWHLSCVEKCPPQITFIAKYATANDKNGNRKLTIRDEQWPELADPTPY DIFGIPKAGSGNPKLDKKSLKKKYHRYVKLYHPDHSDNIQIFSSEKVTNSDSKSPLLLTS SEKLHRFKVISQAYDILCDPKKKIVYDTTRQGWTTSYSPRSNVNTENYQYAGSYGYHSNA QYEYWNAGTWEDANSMKNERIQENINPWTVIGIICGLAICIEGTALLAKIQESLSKAEFT HDESGLHLIQSYTNYGLDTDKFSRLRRFLWFRTWGLYKSKEDLDREAKINEEMIRKLKAA K
JIDl is encoded by a non-essential gene comprising an ORF that is 0.906 kbp in size and is located on chromosome XVI. A published nucleotide coding sequence of JIDl is as follows, although it will be appreciated that the sequence can be modified by degenerate substitutions to obtain alternative nucleotide sequences which encode anidentical proteinproduct:
ATGCTACACCATAAGTTCGTATACCCATTTTTATTCAAGTGGCACTTATCATGTGTAGAA AAGTGTCCCCCACAAATCACTTTTATAGCTAAGTATGCTACAGCGAACGATAAAAATGGC AATAGAAAACTTACGATAAGGGATGAACAATGGCCTGAGTTGGCAGATCCAACTCCCTAT GATATTTTTGGCATTCCAΆΆGGCCGGATCTGGAAATCCTAAACTGGACAΆGAAGTCGTTA AAAAΆAAAΆTATCATCGTTATGTAΆAATTGTACCACCCTGACCATTCCGATAΆCATTCAA ATATTTAGCTCAGAAAAGGTTACCAACAGTGATAGTAΆATCACCGCTGCTGCTAACATCA AGCGAΆAAΆCTACATAGATTTAAΆGTCATCTCTCAAGCATATGATATTCTTTGTGACCCA AAGAAAAAGATCGTATATGACACAACGAGGCAAGGCTGGACCACATCGTATTCACCACGT TCTAACGTTAATACTGAΆΆΆTTACCAATATGCCGGCTCTTATGGCTACCACTCTAACGCG CAGTATGAATACTGGAACGCTGGGACTTGGGAAGACGCAAATAGCATGAAAAACGAAAGA ATTCAAGAAAACATCAΆCCCATGGACCGTTATTGGCATAATTTGTGGCCTAGCTATATGC ATCGAΆGGGACTGCGTTGTTΆGCCAAAATCCAGGAGTCTCTGAGCAΆGGCCGAATTTACT CATGACGAAAGTGGATTACATTTGATTCAGTCATACACGAATTATGGTCTTGATACTGAC AAATTTTCCAGATTGAGGCGGTTCTTATGGTTTAGAACTTGGGGACTTTACAAGTCGAAA GAGGATTTAGATAGAGAAGCCAΆGATCAΆTGAAGAAATGATACGCAAΆCTGAΆΆGCAGCT
AAATGA
Further information on JIDl can be obtained from the URL address http://db.yeastgenome.org/cgi-bin/singlepageformat?sgdid:=S000006265 It will be appreciated that, by "JE)I", we include fragments or variants thereof having equivalent JIDl -like activity.
HLJl is another 51. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the endoplasmic reticulum membrane. A published protein sequence for the protein HIj Ip is as follows:
MSFTEDQEKIALEILSKDKHEFYEILKVDRKATDSEIKKAYRKLAIKLHPDKNSHPKAGE AFKVINRAFEVLSNEEKRSIYDRIGRDPDDRQMPSRGAASGFRGSAGGSPMGGGFEDMFF NSRFGGQRAGPPEDIFDFLFNAGGSPFGASPFGPSASTFSFGGPGGFRVYTNNRGGSPFM RQQPRSRQQQQQAEENAVNSQLKNMLVLFIIFIVLPMIKDYLFS
HLJl is encoded by a non-essential gene comprising an ORP that is 0.675 kbp in size and is located on chromosome XIII. A published nucleotide coding sequence of HLJl is as follows, 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:
ATGTCTTTCACTGAGGATCAAGAAAAAATCGCGCTAGAAATACTGTCAAAAGACAAGCAT GAGTTTTACGAAATTTTGAAGGTAGATAGGAAAGCCACAGATAGTGAGATCAAGAΆGGCA TACAGAAAΆCTAGCAATCAΆATTGCATCCTGATAAAAΆCTCTCATCCAAAAGCGGGAGAA GCTTTCAAAGTAATTAATAGGGCATTTGAAGTACTAAGCAATGAGGAAAAGCGCAGTATT TATGACAGGATAGGTAGGGATCCTGACGATAGACAAATGCCATCCAGAGGTGCTGCTTCA GGGTTCCGAGGAAGTGCAGGTGGGTCTCCAATGGGTGGCGGATTTGAAGACATGTTTTTC AATTCACGTTTCGGTGGTCAAAGAGCTGGACCACCAGAGGACATATTCGACTTTTTGTTC AACGCAGGCGGCAGCCCATTCGGCGCTTCACCATTTGGGCCTTCTGCTTCCACTTTTTCA TTTGGAGGCCCCGGTGGTTTCAGAGTTTATACTAATAATCGTGGTGGCTCACCGTTCATG CGTCAACAACCCCGCTCAAGACAGCAGCAACAACAAGCAGAAGAAΆΆTGCAGTGAΆTTCG CAATTAAAAAATATGCTCGTTCTTTTCATCATCTTTATTGTTCTTCCTATGATTAAAGAT TACCTGTTTAGTTAA
Further information HLJl can be obtained from the URL address http://db.veastgenome.org/cgi-bin/smglepagefommt?sgdid=S000004771 It will be appreciated that, by "HLJl", we include fragments or variants thereof having equivalent HLJl -like activity.
ERJ5 is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial chaperone DnaJ, and is located in the endoplasmic reticulum. A published protein sequence for the protein Erj5p is as follows:
MNGYWKPAL WLGLVSLS YAFTTIETEI FQLQNEI STKYGPDMNFYKFLKLPKLQNSSTK EITKNLRKLSKKYHPDKNPKYRKLYERLNLATQILSNSSNRKIYDYYLQNGFPNYDFHKG GFYFSRMKPKTWFLLAFIWIWNIGQYI ISIIQYRSQRSRIENFISQCKQQDDTNGLGVK QLTFKQHEKDEGKSLVVRFSDVYWEPDGSETLISPDTLDKPSVKNCLFWRIPASVWNMT FGKSVGSAGKEEIITDSKKYDGNQTKKGNKVKKGSAKKGQKKMELPNGKVIYSRK
ERJ5 is encoded by a non-essential gene comprising an ORP that is 0.888 kbp in size and is located on chromosome VI. A published nucleotide coding sequence of ERJ5 is as follows, 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:
ATGAACGGTTACTGGAAACCTGCGTTGGTTGTCCTGGGATTGGTATCTCTATCATATGCT TTTACCACCATTGAAACAGAAATTTTCCAATTACAAAATGAAATAAGTACGAAATATGGC CCAGATATGAACTTCTACAAGTTCTTGAAGTTACCTAAACTGCAGAATTCTAGTACAAAG GAGATTACAAAAAACTTAAGAAAGCTATCCAAGAAGTACCATCCGGATAAGAACCCTAAA TACCGTAAATTGTATGAAAGGTTAAACCTCGCTACTCAAATTCTTTCAAACAGCTCTAAT CGTAAGATTTATGATTATTATCTACAGAATGGCTTTCCAΆACTATGATTTCCATAAGGGT GGTTTTTATTTTTCCAGAATGAAGCCTAAGACTTGGTTCCTGCTGGCCTTTATTTGGATA GTCGTTAATATTGGGCAGTATATCATTTCTATTATTCAATATCGTTCTCAΆAGATCAAGA ATTGAAAACTTCATCAGTCAGTGTAAACAACAGGATGATACCAATGGACTAGGCGTAAAA CAACTAACGTTTAAACAACATGAAAAGGATGAGGGTAAAAGTTTGGTTGTAAGGTTTAGC GATGTCTATGTTGTAGAGCCTGATGGAAGTGAAACACTAATTTCGCCAGATACCTTGGAT AAΆCCTTCAGTAAAGAACTGTTTGTTTTGGAGAATACCTGCTTCGGTTTGGAACATGACG TTTGGCAAATCTGTTGGTAGCGCAGGAAAAGAAGAAATAATAACGGATAGTAAAAAGTAT GATGGTAACCAAACAAAAAAGGGGAACAAAGTAAAAAAGGGTTCTGCAAAGAAAGGCCAA AAGAAAATGGAATTGCCTAACGGTAAAGTGATCTATTCACGTAAATGA Further information ERJ5 can be obtained from the URL address http://db.veastgenome.org/cgi-bin/singlepageforniat?sgdid=S000001937
It will be appreciated that, by "ERJ5", we include fragments or variants thereof having equivalent ERJ5-like activity.
MGEl is another S. cerevisiae helper protein of interest for the present invention and is also known as YGEl. It is one of several homologs of the bacterial GrpE and is located in the mitochondria. A published protein sequence for the protein Mgelp is as follows:
MRAFSAATVRATTRKSFIPMAPRTPFVTPSFTKNVGSMRRMRFYSDEAKSEESKENNEDL TEEQSEIKKLESQLSAKTKEASELKDRLLRSVADFRNLQQVTKKDIQKAKDFALQKFAKD LLESVDNFGHALNAFKEEDLQKSKEISDLYTGVRMTRDVFENTLRKHGIEKLDPLGEPFD PNKHEATFELPQPDKEPGTVFHVQQLGFTLNDRVIRPAKVGIVKGEEN
MGEl is encoded by an essential gene comprising an ORF that is 0.687 kbp in size and is located on chromosome XV. A published nucleotide coding sequence of MGEl is as follows, 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:
ATGAGAGCTTTTTCAGCAGCCACCGTTAGGGCCACAACTAGGAAGTCGTTCATCCCAATG GCACCAAGAACTCCTTTTGTGACTCCATCATTTACAAAGAATGTAGGCTCAATGAGAAGA ATGAGATTTTATTCTGATGAΆGCCAAAΆGTGAAGAΆTCCΆAAGAAAΆCAΆTGAAGATTTG ACTGAΆGAGCAATCAGAAATCAAGAΆATTAGAGAGCCAGTTAAGCGCGAAGACTAAAGAA GCTTCTGAACTCAAGGACAGATTATTAΆGATCTGTGGCAGATTTCAGAAATTTACAACAA GTCACAAAGAAGGATATTCAGAAAGCTAAGGACTTTGCTTTACAGAΆGTTTGCAAΆGGAT TTATTGGAATCTGTAGATAACTTTGGTCATGCTTTGAATGCTTTTAAAGAGGAAGACTTA CAAAAGTCCAAGGAAATTAGTGATTTGTATACAGGGGTTAGAATGACAAGAGATGTTTTT GAAAACACCCTAAGAAAGCACGGTATTGAAAAATTAGACCCATTGGGAGAACCATTTGAT
CCAAATAAΆCACGAAGCAΆCGTTCGAGTTGCCACAACCTGATAΆGGAACCGGGTACTGTT
TTCCATGTACAACAATTAGGTTTCACCTTGAATGACAGAGTTATCAGACCAGCAAAAGTC
GGAATTGTTAAGGGCGAΆGAGAACTAA Further information MGEl can be obtained from the URL address http://db.yeastgenome.org/cgi-biii/singlepageformat?sgdid=S0000Q5758
It will be appreciated that, by "MGEl", we include fragments or variants thereof having equivalent MGEl -like activity.
FESl is another S. cerevisiae helper protein of interest for the present invention. It is one of several homologs of the bacterial GrpE and is located in the cytoplasm. A published protein sequence for the protein Feslp is as follows:
MEKLLQWSIANSQGDKEAMARAGQPDPKLLQQLFGGGGPDDPTLMKESMAVIMNPEVDLE
TKL VAFDNFEMLiENLDNANNiENLKLWEPLLDVL VQTKDEELRAΆALS I IGTAVQNNLD SQNNFMKYDNGLRSLIEIASDKTKPLDVRTKAFYALSNLIRNHKDISEKFFKLNGLDCIA PVLS DNTAKPKLKMRAI ALLTAYLS S VKI DENI I S VLRKDGVI ES T I ECLS DESNLNI I D RVLSFLSHLISSGIKFNEQELHKLNEGYKHIEPLKDRLNEDDYLAVKYVL
FESl is encoded by a non-essential gene comprising an ORF that is 0.873 kbp in size and is located on chromosome II. A published nucleotide coding sequence of FESl is as follows, 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:
ATGGAAAAGCTATTACAGTGGTCTATTGCGAATTCTCAAGGGGACAAAGAAGCTATGGCT AGGGCCGGCCAACCTGATCCTAAATTGCTACAGCAGTTATTCGGTGGTGGTGGTCCTGAC
GATCCAACCTTAATGAAAGAATCCATGGCTGTTATTATGAATCCGGAGGTTGACTTAGAA
ACAAAACTCGTTGCATTTGACAACTTTGAAATGTTGATTGAGAACTTAGATAATGCTAAT
AATATCGAAAATTTAAAACTGTGGGAGCCATTGTTGGATGTTCTTGTTCAGACGAAGGAT
GAAGAACTACGTGCTGCTGCTTTATCCATTATTGGAACGGCTGTGCAAAACAACTTGGAT TCGCAAAATAATTTCATGAAATACGACAATGGTCTGCGAAGCCTTATCGAAATAGCTAGT
GACAAGACAAAGCCACTCGACGTGAGAACAAAAGCTTTTTACGCACTATCTAATCTAATA
AGAAACCACAAAGATATCTCAGAAAAGTTTTTCAAATTAAATGGGCTCGACTGCATAGCA
CCTGTATTAAGTGATAACACCGCCAAACCAAAACTGAAAATGAGAGCCATTGCCTTATTG
ACCGCATATTTGTCATCTGTTAAGATTGATGAAAATATAATCAGTGTGCTGAGAAAGGAT GGAGTAATTGAAAGTACGATTGAGTGCTTGTCTGACGAGAGTAACTTGAACATCATAGAT
AGAGTTCTGTCTTTTCTCTCTCACCTGATATCTTCCGGAATAAAATTTAATGAACAGGAA TTGCACAAΆTTGAACGAΆGGTTACAAACATATCGAGCCTCTAAΆGGACAGACTTAATGAA GACGATTATTTAGCCGTAAAGTATGTATTATGA
Further information FES 1 can be obtained from the URL address http://db.veastgenome.org/cgi-bin/smglepagefoi-mat?sgdid=:S000000305
It will be appreciated that, by "FESl", we include fragments or variants thereof having equivalent FESl -like activity.
Variants and fragments of the above JEMl, LHSl5 SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl5 SSB2, ECMlO5 MDJl5 MDJ2, EROl5 ERV2, EUGl5 MPDl5 MPD2, EPSl, PDIl, DERI, DER3, HRD3, UBC7, D0A4, HACl5 SEC63, YDJl5 XDJl5 APJl5 SISl, DJPl5 ZUOl5 SWA2, JJJl5 JJJ2, JJJ3, CAJl, CWC23, PAM18, JACl5 JIDl, HLJl, ERJ5, MGEl and FESl proteins and encoding polynucleotide sequences, and variants of other naturally occurring JEMl, LHSl5 SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl, SSB2, ECMlO, MDJl5 MDJ25 EROl5 ERV25 EUGl5 MPDl5 MPD2, EPSl5 PDIl5 DERI, DER3, HRD3, UBC7, D0A4, HACl5 SEC63, YDJl5 XDJl5 APJl, SISl, DJPl5 ZUOl5 SWA2, JJJl5 JJJ2, JJJ3, CAJl5 CWC23, PAMl 85 JACl5 JIDl5 HLJl, ERJ5, MGEl and FESlproteins and encoding polynucleotide sequences are also included in the present invention.
A "variant", in the context of a JEMl, LHSl, SCJl5 KAR2, SILl5 FKB2, SSAl, SSA2, SSA3, SSA4, SSEl5 SSE2, SSBl, SSB2, ECMlO5 MDJl5 MDJ2, EROl5 ERV2, EUGl, MPDl5 MPD2, EPSl, PDIl, DERl5 DER3, HRD3, UBC75 D0A4, HACl, SEC63, YDJl5 XDJl5 APJl, SISl5 DJPl, ZUOl5 SWA2, JJJl5 JJJ2, JJJ3, CAJl, CWC23, PAM18, JACl5 JIDl5 HLJl, ERJ5, MGEl or FESl protein, refers to a protein having a sequence as defined above by the present application 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 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, GIy5 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" 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 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 JEMl, LHSl, SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl, MDJ2, EROl3
ERV2, EUGl, MPDl, MPD2, EPSl, PDIl, DERI, DER3, HRD3, UBC7, DOA4,
HACl, SEC63, YDJl, XDJl, APJl, SISl, DJPl, ZUOl, SWA2, JJJl, JJJ2, JJJ3,
CAJl, CWC23, PAM18, JACl, JIDl, HLJl, ERJ5, MGEl and FESl proteins, refers to a protein wherein at one or more positions there have been deletions. Thus the fragment may 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 protein as defined above. Particularly preferred fragments of a protein comprise one or more whole domains of the desired protein.
A fragment or variant of a JEMl, LHSl, SCJl, KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl, MDJ2, EROl, ERV2,
EUGl, MPDl, MPD2, EPSl, PDIl, DERI, DER3, HRD3, UBC7, DOA4, HACl, SEC63, YDJl, XDJl5 APJl, SISl, DJPl, ZUOl5 SWA2, JJJl5 JJJ2, JJJ35 CAJl, CWC23, PAMl 8, JACl, JIDl, HLJl5 ERJ5, MGEl or FESl protein may be a protein that, when expressed recombinantly in a host cell, can complement the deletion of the same endogenously encoded gene in the host cell, such as S. cerevisiae, and may or may not, for example, be a naturally occurring homolog of the protein upon which it is based, such as a homolog encoded by another organism, such as another yeast or other fungi, or another eukaryote such as a human or other vertebrate, or animal or by a plant.
A fragment or a variant of a polynucleotide encoding a JEMl5 LHSl, SCJl5 KAR2, SILl, FKB2, SSAl, SSA2, SSA3, SSA4, SSEl, SSE2, SSBl, SSB2, ECMlO, MDJl, MDJ2, EROl, ERV2, EUGl, MPDl, MPD2, EPSl, PDIl, DERI, DER3, HRD3, UBC7, D0A4, HACl, SEC63, YDJl, XDJl, APJl, SISl, DJPl, ZUOl, SWA2, JJJl5 JJJ2, JJJ3, CAJl5 CWC23, PAM18, JACl5 JIDl, HLJl, ERJ5, MGEl or FESl protein may be a polynucleotide that comprises a sequence that encodes a fragment or variant of the protein as defined above.
The present invention will now be exemplified with reference to the following non-limiting examples and figures.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1 to 9, 11 to 16, 21, 23-25 and 28 show various plasmid maps as described in the following examples.
Figure 10 shows analysis of HACl splicing at log phase by qRT-PCR in the strain AH22 (ura3) [pAYE329]. Helper protein overexpression plasmids are shown on the x-axis. Data are normalised to ACTl transcript levels and presented as fold changes from AH22 (ura3) [pAYE329, YCplac33]. All values shown represent duplicate analysis of mRNA levels from single experimental cultures.
Figure 17 shows SDS-PAGE gels for quantification of rHA production in overexpression strains. Sample labels shown indicate overexpression plasmids transformed into the strain AH22 (ura3) [pAYE329]. Duplicate samples represent two independent shake flasks from the same transformant.
Figure 18 shows quantification of main rHA band in transformed and control strains, by analysis of SDS-PAGE gel of Fig. 17 using densitometry. Values are normalised (based on culture optical density readings) to account for different growth rates observed between strains.
Figure 19 shows quantification of main rHA band in transformed and control strains, by analysis of SDS-PAGE gel of Fig. 17 using densitometry, expressed as a percentage of determined rHA production by the negative control YCplac33. Values are normalised (based on culture optical density readings) to account for different growth rates observed between strains.
Figure 20 shows quantification of rHA fragments relative to total rHA, by analysis of SDS-PAGE gel of Fig. 17 using densitometry, expressed as a percentage of detected rHA fragments relative to total rHA levels detected (total rHA = full length rHA + degradation products). Values are normalised (based on culture optical density readings) to account for different growth rates observed between strains.
Figure 22 shows a comparison of recombinant transferrin titres by rocket immunoelectrophoresis. A = Control Strain [pDB3213]; B = Control Strain (ura3) [pTPC17 pDB3213]. Duplicate 1OmL shake flasks cultures were inoculated with yeast and incubated with shaking at 200rpm for 4-days at 300C. 5μL culture supernatant loaded per well of a rocket immunoelectrophoresis gel. Plasma Tf standards concentrations are in μg/mL. 20μL goat anti-Tf / 5OmL agarose. Precipin was stained with Coomassie blue.
Figure 26 shows the effect of LHSl, JEMl and SILl co-expression on rHA production, when rHA is fused to different leader sequences. Two separate transformants for each strain were inoculated into 5OmL shake flasks containing 1OmL BMMD and incubated with shaking at 200rpm for 4-days at 300C. 20μL of culture supernatant was loaded per well of a 4-12% SDS-PAGE gel and run for 50mins in MOPS buffer. Gel A shows the results obtained with plasmid pDB2244, which encodes a ESAJMFa-I fusion leader sequence (A = AH22 (ura3) [pDB2244 YCplac33]; B = AH22 (ura3) [pDB2244 pTPC17]). Gel B shows the results obtained with plasmid pDB2286, which encodes an invertase leader sequence (C = AH22 (ura3) [pDB2286 YCplac33]; D = AH22 (ura3) [pDB2286 plPCl 7]). Gel C shows the results obtained with plasmid pDB2287, which encodes the MFa- 1 leader sequence (E = AH22 (ura3) [pDB2287 YCplac33]; F = AH22 (ura3) [pDB2287 pTPC17]).
Figure 26, part D, shows densitometric quantification of rHA secretion. Gels shown in Figure 26 A-C were analysed by densitometry and comparison to rHA standard curves. Data presented above represents quantification of single rHA bands. For each strain two transformants were analysed (samples A and B in Fig. 26D).
Figure 27 shows the DNA sequence of the human GM-CSF cDNA with an incorporated N-terminal Met codon.
Figure 29 A shows an SDS-PAGE gel for quantification of GM-CSF production.
Lanes 2-5 show GM-CSF production in the control strain [uraS) [pDB2109
YCplac33]. Lanes 6-9 show GM-CSF production in the control strain (ura3)
[pDB2109 pTPC17.
Figure 29 B shows the results of densitometric analysis of the SDS-PAGE gel shown in Figure 29 A, as further given in Table 9, below.
EXAMPLE l
A strain of S. cerevisioe that possesses increased production of a recombinant protein was produced by the following methodology. Strains. The S. cerevisiae strain used was a histidine revertant of AH22 (cir° a leu2-3 Ieu2-112 Ms4 canR). AH22 is further described in Mead et al, 1986, MoI. Gen. Genet, 205, 417-421. A polynucleotide encoding a recombinant heterologous protein expression cassette was introduced by S. cerevisiae transformation performed according to Ito, H., et al. (Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 153, 163-168, (1983)).
Media. Yeast strains were grown in rich broth medium, YEP (1% yeast extract 2% w/v Bactopeptone).
Protein assays. Yeast cells were grown in 10 ml cultures for 72 hours to a density of 5 x 107 cells/mL at 3O0C in YEP 2% (w/v) sucrose. In order to analyse the soluble heterologous protein fraction of yeast, cells were harvested by centrifugation and disrupted in phosphate buffered saline by vortexing with 40 mesh glass beads. The soluble fraction was collected as the supernatant of a 10,000 x g centrifugation. The fraction was assayed for the presence of heterologous protein by polyacrylamide gel electrophoresis and Western blot, using appropriate commercially available antibodies.
Mutagenesis. Yeast cells to be mutated were grown in 100 ml defined medium (0.65% (w/v) YNB; 2% (w/v) sucrose; Na2HPO4/citric acid pH 6.5) to OD650 = 0.5. Cells were harvested by centrifugation and resuspended in 100 ml defined medium. To 2 ml of washed cells was added 10 microlitres, 20 microlitres, 40 microlitres, 80 microlitres or 160 microlitres of the mutagen stock solution. The cells were then incubated at 3O0C, 200 rpm for 30 min. One ml of mutated cells was washed twice with 1 ml sterile distilled water and finally resuspended in 1 ml YEP. The percentage of cells that survived the mutagenic treatment was assessed by spreading an aliquot of each mutagenic reaction onto YEP, 2% (w/v) sucrose plates. Mutagen stock solutions were prepared as follows. N-methyl-N-nitro-N- nitrosoguanidine (NTG) was dissolved in ethanol at 5 mg/mL; 4 nitroquinoline N- oxide (NQO) was resuspended in acetone at 10 mg/mL and then diluted 1 in 100 to 0.1 mg/mL with K2HPO4ZKH2PO4 (pH 7.0); 1, 2, 7, 8-diepoxyoctane (DEO) and ethyl methanesulphonate (EMS) were both supplied as liquids (Sigma) and were used without dilution.
After mutagenesis, a S. cerevisiae strain was identified with a higher level of production of a recombinant protein, compared to its ancestral strain (data not shown).
EXAMPLE 2
The expression of genes in the strain identified in Example 1 was compared to the expression of genes in the ancestral strain from which it was derived (i.e. the ancestral strain displays lower levels of production of a recombinant protein).
The comparison was made by using microarray analysis. Yeast cells to be analysed were grown in 100 ml defined medium (0.65% (w/v) YNB; 2% (w/v) dextrose; Na2HPO4/citric acid pH 6.5) to OD600 = 2.0. The cells were immediately harvested by centrifugation and frozen by immersion in liquid nitrogen. KNA suitable for microarray analysis was prepared by disruption of the cells using a micro dismembrator (Braun Melsungen, Germany) all as described by Jones et al, 2003, Physiol Genomics, 16, 107-118. cDNA synthesis, labelling, hybridisation to high-density oligonucleotide arrays (Affymetrix - Yeast S98) and scanning were carried out as described by protocols provided by the manufacturer (Affymetrix Inc, USA). The subsequent data was analysed using the MAS 5.1 and DTM 3.0 software programs (Affymetrix Inc, USA).
Genes identified as being up-regulated in the strain identified in Example 1, compared to the ancestral strain, include - Table 1
Figure imgf000243_0002
Figure imgf000243_0001
It will be recognised that none of SSAl1 SSA2, SSEl, SSBl, SSB2, MDJl or MDJ2 were identified as being over-expressed in the strain identified in Example 1. However, these helper proteins have been included in the present invention as a result of their functional association to the helper proteins whose genes have been identified as being upregulated in the strain isolated in Example 1. For example, the genes encoding SSA3, SSA4 and SSB2 have all been identified as being over- expressed; SSAl, SSA2, SSEl, SSBl and SSB2 are functional equivalents of these helper proteins and so it is anticipated that over-expression of the genes encoding any of SSAl, SSA2, SSEl, SSBl or SSB2 would cause the same phenotype as the over-expression of the genes encoding any of SSA3, SSA4 or SSB2. Similarly the gene encoding ECMlO has been identified as being over-expressed; MDJl and MDJ2 are functional equivalents of ECMlO and so it is anticipated that the over- expression of either of the genes encoding MDJl or MDJ2 would cause the same phenotype as the over-expression of the gene encoding ECMlO. EXAMPLE 3
The example describes the vector construction and yeast transformation for the overexpression of the representative helper proteins LHSl, SLSl, JEMl and SCJl.
Table 2: Primers used
Figure imgf000244_0001
Figure imgf000245_0001
pBST HO regions: HO regions were amplified by PCR from BY4741 (Brachmann et al., 1998, Yeast, 30; 14(2): 115-32) genomic DNA using the primers shown in Table 2. Fast Start High Fidelity PCR system (Roche) was used with the conditions as recommended: 50μL final volume containing 0.2mM dNTPs, 1.8mM MgCl2, 0.4μM forward and reverse primers, lOOng template genomic DNA, 2.5U polymerase and H2O to volume. Cycling conditions: 95°C for 2 rnins followed by 35 cycles of 950C 30s, 60°C 30s, 72°C 1 min and 720C 7 mins for final elongation.
Fragments were gel extracted from a l%(w/v) agarose TAE gel using the
GeneClean III kit (Q-bio Gene). Purified DNA was digested with the appropriate enzymes, Notl and Mlul for HO 5' region, MwI and Claϊ for HO 3' region. ρBST+ (WO99/00504) was digested with Notl and CM. Fragments were purified as above. A three way ligation was performed using a Rapid Ligation Kit (Roche) as per manufacturers instructions. Ligations were transformed into the E. coli strain DH5α. Diagnostic restriction digests were performed on mini-prep DNA to confirm the ligation was successful. The plasmid map is shown in Figure 1.
Polylinkers: To facilitate the cloning of the helper genes a polynucleotide linkers were incorporated into pBST + HO regions (Figure 1) and into YCplac33 (Gietz and Sugino, 1988, Gene, 74, 527-534).
Complementary single stranded oligonucleotides were annealed as follows: lμL of a lOOμM solution of each oligo (Poly For and Poly Rev, Table 2) was added into a 50μL total volume containing 1Ox restriction buffer (Roche Buffer H for pBST HO polylinker, Buffer B for YCplac33 polylinker). Samples were placed into a PCR machine and heated to 98°C for 4 mins. Samples were then held for 1 min with the temperature dropping 1°C every cycle down to 30°C. The annealed polylinkers were then digested by addition of the appropriate restriction enzyme (MuI, EcόRl for pBST HO polylinker, BamHL, EcoBl for YCplac33 polylinker). Digested polylinkers were gel extracted as previously and ligated into the corresponding vector digests. Incorporation of polylinkers was confirmed by linearising plasmids with all restriction sites present in polylinkers. Vectors produced are shown as Figures 2 and 3 respectively.
Production of promoter/open reading frame constructs: All four open reading frames (ORFs) and promoters were amplified by PCR, from the genomic DNA of an AH22 derivative, using Vent polymerase (NEB). Reactions were setup as per manufacturers instructions with an annealing temperature of 50° C. All fragments were gel extracted and resuspended in 5μL of water. lμL was run on gel to check fragment presence and quantity. Promoters and ORFs were joined according to the method of Shevchuk et al (Nucleic Acids Res., 2004, 32(2), el9.). lOOng of ORP and an equimolar amount of promoter was used in the first PCR stage. lOμL from this was used in the second PCR stage. Primers were added to a final concentration of 0.4μM.
Second stage PCRs were run on a l%(w/v) agarose TAE gel and bands extracted of the expected size (promoter + ORF length). Extracted fragments were A-tailed using Fast Start High Fidelity polymerase (Roche) and cloned into the Topo pCR2.1 vector (Invitrogen). Plasmid DNA was restriction digested to confirm the correct insert and subsequently sequenced.
Assembly of overexpression constructs: Restriction digests were performed to release promoter/ORF constructs from Topo pCR2.1 vectors. Fragments were gel extracted and ligated into the pBST HO polylinker vector, digested accordingly. In the first instance, constructs were produced containing each individual promoter/ORF and containing all four. This required subsequent rounds of plasmid transformation, digestion and ligation. The vector containing all four promoter/ORFs is shown in Figure 4.
For insertion of promoter/ORF constructs into the centromeric vector, YCplac33 polylinker, a PmellAlel digest was performed on pBST HO POLY (Figure 4) containing the required promoter/ORFs, and YCplac33 polylinker. The fragment released from pBST HO POLY was ligated with the digested YCplac33 polylinker vector. The vector containing all four promoter/ORFs is shown in Figure 5.
Insertion of URAS marker into pBST HO POLY: The URA3 marker was amplified by PCR from the vector YCp50 (Rose et al, 1987, Gene, 60, 237-243) using Fast Start High Fidelity polymerase (Roche) with an annealing temperature of 50°C. The fragment was gel extracted, digested with PacVPmel and ligated into each pBST HO POLY vector containing the required promoter/ORFs (also PacVPmel digested). It is important the URA3 fragment be introduced last as it contains sites for restriction enzymes used elsewhere in construction of the plasmid. The vector produced containing all four promoter/ORFs is shown in
Figure 6.
Chromosomal integration: The helper gene constructs were integrated into the genome of a S. cerevisiae host cell as follows. The vector pBST HO POLY URA3 COMP (Figure 6) was digested with Notl and Sacll. Approximately 2-3 μg of the required fragment was gel extracted and used to transform a ura3 derivative of AH22 [pAYE329] using a yeast transformation ldt (Sigma). Transformations were plated onto minimal media and incubated at 30°C until colonies appeared. The construction of plasmid pAYE329 is described in Sleep et al, 1990, Gene, 101, 89-96. A ura3 auxotrophic mutant of the AH22 derivative was created by 5- fmoro-orotic acid selection as described by Boeke et al, 1987, Methods Enzymol, 154, 164-175.
Alternatively, the helper gene constructs may be introduced on a centromeric vector. For the YCplac33 based-vectors, 500ng of plasmid DNA may be used to transform a S. cerevisiae host cell as above.
EXAMPLE 4
This example describes a modified protocol for vector construction and yeast transformation for the overexpression of the representative helper proteins LHSl, SILl, JEMl and SCJl.
Table 3: Primers used
Figure imgf000248_0001
Figure imgf000249_0001
Construction of pTPAO 1 : 5' and 3' regions of the HO open reading frame were amplified by PCR from BY4741 (Brachmann et al., 1998, Yeast, 30;14(2):l 15-32) genomic DNA using the primers AOl -02 (5') and A03-04 (3'). Fast Start High Fidelity PCR system (Roche) was used with the conditions as recommended, as defined in Example 3, above.
Fragments were gel extracted from a l%(w/v) agarose TAE gel using the GeneClean III kit (Q-bio Gene). Purified DNA was digested with the appropriate enzymes, Notl and MwI for HO 5' region, MwI and CM for HO 3' region. ρBST+ (WO99/00504) was digested with Not! and CM. Fragments were purified as above. A three-way ligation was performed using a Rapid Ligation Kit (Roche) as per manufacturers instructions. Ligations were transformed into the E. coli strain DH5α. Diagnostic restriction digests were performed on mini-prep DNA to confirm the ligation was successful. The plasmid map of TPAOl is shown in Figure 7.
Polylinkers: To facilitate the cloning of the helper genes a polynucleotide linker was incorporated into pTPAOl (Figure 7) and into YCplac33 (Gietz and Sugino, 1988, Gene, 74, 527-534).
Complementary single stranded oligonucleotides were annealed as follows: lμL of a lOOμM solution of each oligo (A05-06 and A13-14) was added into a 50μL total volume containing 10x restriction buffer (Roche Buffer H for pTPAOl polylinker, Buffer B for YCplac33 polylinker). Samples were placed into a PCR machine and heated to 98°C for 4 mins. Samples were then held for 1 min with the temperature dropping TC every cycle down to 30°C. The annealed polylinkers were then digested by addition of the appropriate restriction enzyme (MIuI, EcάRl for pTPAOl polylinker, BamBI, Eco~Rl for YCplac33 polylinker). Digested polylinkers were gel extracted as previously and ligated into the corresponding vector digests. Incorporation of polylinkers was confirmed by linearising plasmids with all restriction sites present in polylinkers. Vectors produced are shown as Figures 8 and 11 respectively.
Production of promoter/open reading frame constructs: LHSl, SILl, JEMl and SCJl open reading frames (ORFs) plus approximately 300bp of terminator sequence (3' of ORF) and promoters were amplified by PCR, from the genomic
DNA of an AH22 derivative, using Vent polymerase (NEB) (see Table 3 for primers used). Reactions were setup as per manufacturers instructions with an annealing temperature of 50° C. AU fragments were gel extracted and resuspended in 5μL of water. lμL was run on a gel to check fragment presence and quantity.
Promoters and ORFs for LHSl and SCJl were joined according to the method of Shevchuk et al {Nucleic Acids Res., 2004, 32(2), el9.). lOOng of the ORF fragment and an equimolar amount of promoter fragment was used in the first PCR stage. lOμL from this was used in the second PCR stage. Primers were added to a final concentration of 0.4μM.
Second stage PCRs were run on a l%(w/v) agarose TAE gel and bands extracted of the expected size (promoter + ORF + terminator). Extracted fragments were A- tailed using Fast Start High Fidelity polymerase (Roche) and cloned into the TOPO pCR2.1 vector (Invitrogen). Plasmid DNA was restriction digested to confirm the correct insert.
Promoters and ORFs for <S7ZJ and JEMl were digested with restriction enzymes corresponding to sites incorporated into primers used for PCR (see Table 3). Promoter and ORF fragments were then joined by three way ligation with digested pTPA02.
The ACTl promoter and terminator were amplified by PCR from the genomic DNA of an AH22 derivative and gel extracted. Purified fragments. were digested with restriction enzymes corresponding to sites incorporated into primers used for PCR and ligated in a three way ligation with PacllPmel digested pTPA02 to create pTPA03 (Figure 9).
The HACl ORF was amplified by PCR from cDNA derived from RNA from an AH22 derivative treated with the reducing agent dithiothreitol (DTT). The spliced form of HACl (HACl') was identified as a 717bp fragment and gel extracted. The extracted fragment was then digested with Xbal and ligated into pTPA03 digested with the same enzyme. Diagnostic restriction digests were used to confirm that the
HACl ORF was present in the correct orientation relative to the ACTl promoter and terminator sequences. The resultant plasmid pTPCOl is shown in Figure 13.
All ORFs were sequenced and, with exception of LHSl, were shown to contain the same sequence as that published for the strain S288C. Repeat sequencing of multiple cloned PCR products for LHSl confirmed that the AH22 derived clones contained a single base change from the S288C sequence. The base change at position 1215 (relative to the first base of the start codon) results in a change from A to C, which produces a Lys to Asn substitution at position 405.
Assembly of overexpression constructs: Restriction digests (see Table 3) were performed to release promoter/ORF constructs from TOPO pCR2.1 vectors.
Fragments were gel extracted and ligated into the pTPA02 vector, digested accordingly. In the first instance, constructs were produced containing each individual promoter/ORF and then containing all four. This required subsequent rounds of plasmid transformation, digestion and ligation. The vector containing all four promoter/ORFs is shown in Figure 12.
For insertion of the various promoter/ORF constructs (with the exception of HACl) into the centromeric vector, pTPA05 (Figure 11), an AleVXhόl digest was performed on the various pTPA02 based vectors containing the required promoter/ORFs (e.g. pTPC08 (Figure 12) for LHSl, SILl, JEMl and SCJl), and an AIeIISaK digest on pTPA05 (Figure 11). The various promoter/ORF fragments released were ligated into AIeUSaFL digested pTPA05 to create a series of vectors including pTPC 18 (Figure 14) containing all four promoter/ORFs. Plasmid pTPC17 (Example 4, Figure 15) contained the LHSl, SILl and JEMl ORFs expressed from YCplac33. pTPC17 was constructed by cloning an approximately 9.0-kb Alel-Xhol DNA fragment from ρTPC07 (Figure 16) that contained the expression cassette for the LHSl, SILl and JEMl ORFs, into pTPA05 (Figure 11) which had been digested with^4/el and Sail. The expression cassette for the LHSl, SILl and JEMl ORFs was assembled in pTPA05 in a similar method to that described for pTPC08 (Figure 12), but using the promoter/ORF constructs from TOPO pCR2.1 vectors for LHSl, SILl and JEMl expression.
For insertion of the HACl promoter/ORF (Figure 13) into the centromeric vector pTPA05, an AIeIlBcR digest was performed on pTPCOl (Figure 13) and an AleVBamHI digest was performed on pTPA05 (Figure 11). The HACl AIeVBcR fragment released from pTPCOl was ligated into the Alel/BamΗI digested pTPA05.
The various promoter/ORF constructs comprising the YCplac33 based plasmids P1TPCIl, pTPC12, ρTPC13, pTPC14, pTPC15, pTPC17 and pTPC18 are shown in Table 4.
Table 4: Plasmid compositions
Figure imgf000253_0001
Insertion of URA3 marker into pTPA02: The UJRA3 marker was amplified by PCR from the vector YCp50 as described above in Example 3. The fragment was gel extracted, digested with Pacl/Pmel and ligated into each pTPA02 based vector containing the required promoter/ORFs (also Pacl/Pmel digested). It is important the URA3 fragment be introduced last as it contains sites for restriction enzymes used elsewhere in construction of the plasmid.
Chromosomal integration: The helper gene constructs were integrated into the genome of a S. cerevisiae host cell by digestion of the vector pTPCOS (Figure 12) with Notl and Sacll and transformation of a ura3 derivative of AH22 [pAYE329] as described in Example 3, above.
Alternatively, the helper gene constructs may be introduced on a centromeric vector. For the YCplac33 based-vectors, 500ng of plasmid DNA may be used to transform a S. cerevisiae host cell as above.
EXAMPLE 5
Plasmids constructs were produced for the overexpression of the genes LHSl, JEMl, SCJl and SlLl as described in Example 4, above.
The spliced form of the transcription factor HACl (referred to as HACl1) was also overexpressed using the vector series produced. Due to the regulatory role of HACl within the unfolded protein response, HACIs was overexpressed alone, not in conjunction with the other chaperone genes described here.
All genes were overexpressed from YCplac33 based vectors (Table 4) and transformed into the ura3 auxotrophic mutant of the ancestral S. cerevisiae strain (a histidine revertant of AH22) [pAYE329] defined ha Example 4, above.
Overexpression was confirmed using real time PCR. Taqman hybridisation probes were designed to bind specifically to each gene under investigation plus ACTl, used here as an endogenous control. An additional probe was designed for the gene HACl to bind across the exon-exon junction - resulting in binding only to the spliced form. The proportion of HACl1 relative to total HACl can thus be determined.
Table 5: Taqman probe/primer sequences and binding co-ordinates
Figure imgf000255_0001
* means probe binding coordinates, relative to start codon The relative standard curve method of transcript quantification was used as described by Applied Biosystems in the 'ABI PRISM 770 Sequence Detection System : User Bulletin #2' document. This can be downloaded from the Applied Biosystems' website (www.appliedbiosystems.com). Equivalent technical disclosure of a suitable quantitative RT-PCR method can be found in Bustin, 2000, Journal of Molecular Endocrinology, 25, 169-193. This method allows quantification of the gene of interest relative to an endogenous control genethat is known to exhibit constant expression across experimental conditions.
All real time PCR was carried out on cDNA derived from RNA extracted from log phase (OD6O0 = 2) BMMD yeast cultures. Overexpression was assessed by comparison of strains with a control yeast strain transformed with the base vector YCplac33 and are expressed as fold changes.
Table 6: Summary of overexpression levels achieved
Figure imgf000256_0001
As shown below in Table 6, overexpression levels vary between the different constructs. Levels achieved range from 2.03 fold for SILl to 22.63 fold for LHSl.
The effect of overexpression of HACl'', LHSl, JEMl, SlLl and SCJl on the induction of the stress-related unfolded protein response (UPR) in a host cell was investigated by measuring the levels of HACl1 and total HACl transcript levels in AH22 (ura3) [ρAYE329] host cells transformed with Ycplac33 (as a negative control), pTPCl 1, pTPC12, pTPCIS, pTPCH, pTPC15 or pTPC18. Total HACl transcript levels are the sum of HACl1 transcript levels and unspliced HACl transcript levels. A reduced proportion of the level of HACl1 transcript levels compared to total HACl transcript levels is indicative of reduced stress and reduced UPR signalling.
Figure 10 shows that individual over-expression of LHSl (pTPCIS) or JEMl (pTPC14) or simultaneous over-expression of all of LHSl, JEMl5 SILl and SCJl (pTPC18) resulted a reduced proportion of the level of HACl1 transcript levels (compared to total HACl transcript levels) compared to the control. This indicates that over-expression of the above-identified helper proteins can help to reduce stress in cultured cells and avoid the unnecessary induction of the UPR.
EXAMPLE 6
The levels of recombinant protein production achieved by the transformed strains described in Examples 4 and 5 (see Table 4), above, were analysed. In this case, the recombinant protein was recombinant human albumin ("rHA") expressed from the plasmid pAYE329, described in Sleep et al, 1990, Gene, 101, 89-96.
All analysis was performed on cultures grown for 5 days at 30°C, 200rpm.
Culture supernatants were run immediately on gels to prevent any rHA proteolysis/degradation that could otherwise occur during freezing and overnight storage at -2O0C. Each of the three bands (main rHA band plus two degradation products) were quantified by densitometry. This gives an indication of rHA production levels and the level of proteolysis occurring in each strain. The mutagenised strain identified in Example 1 was also included as a positive control.
Results of the analysis are shown in Figure 17. It is apparent from a comparison of the results for the ancestral strain expressing recombinant albumin from pAYE329/YCplac33 ("YCplac33") and the mutagenised strain identified in Example 1 as possessing increased recombinant protein production ("+ve control") that the niutagenised strain is not only capable of producing increased levels of rHA, but additionally displays reduced levels of rHA degradation compared to the ancestral strain. Moreover, Figure 17 is particularly clear in demonstrating that strain transformed with pTPC17 (i.e. the ancestral strain transformed to over-express LHSl, JEMl and 57Xi) also displays reduced levels of rHA degradation compared to the untransformed ancestral strain.
Further characterisation of the effect of the defined transformations is possible in view of the analysis of the SDS-PAGE gel by densitometry, the results of which are present in Table 7, below, and Figures 18 and 19.
Table 7 : Comparison of rHA levels, as percentage of YCplac33 control production levels. In the third column, the rHA production levels have been normalised (based on culture optical density readings) to account for different growth rates observed between transformants.
Figure imgf000258_0001
Table 7, above, and Figures 18 and 19, show that the individual overexpression of HACl, LHSl, JEMl, SJLl and SCJl results in an increase in rHA production, on a per cell basis (i.e. when results are normalised by culture OD). However, the negative growth effect of SCJl overexpression resulted in an overall reduction of rHA production on a per culture basis (i.e. when results are not normalised by culture OD).
The overexpression of JEMl alone had the largest measured effect on rHA production.
However, as will be apparent from Figure 17, the strains that individually expressed HACl, LHSl, JEMl, SILl and SCJl still demonstrated relatively high levels of rHA degradation, comparable to the ancestral strain and higher than the mutagenised strain identified in Example 1. By contrast, cells that simultaneously over-express ZHSi, JEMl and SILl demonstrate increased rΗA productivity and a concomitant reduction in rΗA degradation, comparable with the mutagenised strain identified in Example 1. This is further demonstrated in Figure 20. In fact, Figure 20 shows that several of the strains tested show lower levels of degradation compared to the ancestral strain, but this reduction is particularly pronounced in strain transformed with pTPC17.
EXAMPLE 7
This example describes the increased secretion of a recombinant transferrin mutant by over-expression of LHSl, JEMl and 57Zi from the centromeric vector pTPC17 in a Saccharomyces cerevisiae strain containing a 2-micron plasmid encoding the PDIl gene.
A S. cerevisiae strain, the "control strain" as used in WO 2005/061718 and WO 2005/061719 was used to generate a ura3 mutant derivative, referred to herein as "control strain (ura3)" by random mutagenesis and selection on 5-fluoro-orotic acid plates (Boeke et al, 1984, op. cit).
The S. cerevisiae control strain was transformed to leucine prototrophy with pDB3213 (Figure 21) and the control strain (ura3) was co-transformed to both leucine and uracil prototrophy with plasmids pTPC17 (Figure 15) and pDB3213.
Transformation was by a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2 (Elble, R, 1992, Biotechniques, 13, 18- 20; Ito et al, 1983, op. cit.). Transformants were selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates.
The construction of pTPCl 7 is described in Example 4.
Plasmid pDB3213 is similar to pDB2929 (WO 2005/061718, Example 1 and Figure 12), and contains a Notl expression cassette for a non-glycosylated transferrin cloned into pDB2690 (WO 2005/061718, Example 1 and Figure 6). The Notl expression cassette of pDB3213 contains an alternative codon for Leucine-505 in mature transferrin that is the CTG codon (11% codon usage in S. cerevisiae) compared to the CTC codon (6% codon usage in S. cerevisiae) present in pDB2929, a KEX2-wdepea.deat leader sequence (derived from the HSA-pre leader sequence) and mutations within the N-linked glycosylation sites (-N-X-S/T-) that prevent glycosylation of residues N413 and N611.
Transformants of each strain were inoculated into 1OmL BMMD and 1OmL YEPD in 5OmL shake flasks and incubated in an orbital shaker at 300C, 200rpm for 4- days. Culture supernatants were harvested and the recombinant transferrin titres compared by rocket Immunoelectrophoresis (Figure 22). The results indicated that the recombinant transferrin titres in supernatants of both the YEPD and BMMD shake flask cultures were higher when pTPC17 was present. Furthermore, in high cell density fed batch fermentation the recombinant transferrin titres from control strain (ura3) [pTPC17 pDB3213] was 1.7g/L compared to only 0.9g/L for control strain [pDB3213]. Therefore, over- expression of LHSl, JEMl and SILl from the centromeric plasmid pTPC17 had approximately doubled the quantity of the recombinant transferrin product secreted from the S. cerevisiae strain during fermentation.
It is to be noted that pDB3213 encodes an additional copy of PDIl, and these results suggest that over-expression of PDIl (and variants thereof) in conjunction with one, two or all three of LHSl, JEMl, and SILl (e.g. LHSl alone; JEMl alone; SILl alone; LHSl and JEMl; LHSl, and SZLi; JEMl, and SILl; or LHSl, JEMl, and SILl) provide unexpected benefits to the production of a desired protein product.
EXAMPLE 8
This example shows increased secretion of recombinant albumin ("rHA") by over- expression of LHSl, JEMl and SILl from the centromeric vector pTPC17 in a Saccharomyces cerevisiae strain.
Construction of plasmid pDB2243 containing the Notl rHA expression cassette, incorporating the HS AJMFa-I fusion leader sequence, as taught in WO 90/01063, is described in WO 00/44772 (see WO 00/44772, Figure 6). The rHA expression disintegration vector pDB2244 (Figure 23) was created by ligating the Notl expression cassette from pDB2243 into Notl cut pSAC35 (Sleep et al, 1991, Bio/Technology 9, 183-187 and EP 431 880) to generate the plasmid pDB2244 in which the direction of rHA transcription is in the same orientation as that of the LEU2 gene as described in WO 00/44772.
Construction of plasmid pDB2283 containing a Notl rHA expression cassette, incorporating the inyertase leader sequence, was accomplished by replacing the 1.21-kb Bfi'l-Xbal fragment in pDB2243, comprising the HS AJMFa-I fusion leader sequence and part of the human albumin cDNA, with a 1.07-kb blunt end- Xbal fragment from mpl9.7 (EP-A-248 637) and a synthetic double stranded oligonucleotide linker of the following structure —
1 gagtccaatt agcttcatcg ccaataaaaa aacaagctaa acctaattct ctcaggttaa tcgaagtagc ggttattttt ttgttcgatt tggattaaga
Hindi I I -+-—.-
51 aacaagcaaa gatgaagtgg gtaagcttaa cctaattcta acaagcaaag ttgttcgttt ctacttcacc cattcgaatt ggattaagat tgttcgtttc
101 atgcttttgc aagccttcct tttccttttg gctggttttg cagccaaaat tacgaaaacg ttcggaagga aaaggaaaac cgaccaaaac gtcggtttta » Invertase > m i l q a f l f l l a g f a a k
151 atctgca tagacgt
> . . . . » Invertase i s a
which was formed by annealing two complementary single stranded oligonucleotides with the sequences
• 5'TTAAGAGTCCAATTAGCTTCATCGCCAATAAAAAAACAAGCTAAACCT AATTCTAACAAGCAAAGATGAAGTGGGTAAGCTTAACCTAATTCTAACAA GCAAAGATGCTTTTGCAAGCCTTCCTTTTCCTTTTGGCTGGTTTTGCAGC CAAAATATCTGCA3 ' ; and
• 5 ' TGCAGATATTTTGGCTGCAAAACCAGCCAAAAGGAAAAGGAAGGCTTG CAAAAGCATCTTTGCTTGTTAGAATTAGGTTAAGCTTACCCACTTCATCT TTGCTTGTTAGAATTAGGTTTAGCTTGTTTTTTTATTGGCGATGAAGCTA ATTGGACTC3' .
Plasmid mpl9.7 (EP-A-248 637) was digested to completion with Xhol, phenol/chloroform extracted and ethanol precipitated. The recovered DNA was then blunt ended with the Klenow fragment of E. coli DNA polymerase I to remove the Xhol overhang, phenol/chloroform extracted, and ethanol precipitated. The recovered DNA was digested to completion with Xbal. The digestion products were resolved by agarose gel electrophoresis and the 1.07-kb blunt end- Xbal mpl9.7 fragment recovered using the GeneClean III kit (Q-bio Gene).
The rHA expression disintegration vector pDB2286 (Figure 24) was created by ligating the Not! expression cassette from pDB2283 into Notl cut pSAC35 (Sleep et al, 1991, Bio/Technology 9, 183-187 and EP 431 880). Construction of plasmid pDB2284 containing a Notl rHA expression cassette, incorporating the MFa-I leader sequence, was accomplished by replacing the 1.21-kb Bfi'l-Xbal fragment in pDB2243, comprising the KSAJMFa-I fusion leader sequence and part of the human albumin cDNA, with a 1.07-kb blunt end- Xbal fragment from mpl9.7 (EP-A-248637) and a synthetic double stranded phosphorylated oligonucleotide linlcer of the structure -
1 ttaagagtcc aattagcttc atcgccaata aaaaaacaaa ctaaacctaa ctcagg ttaatcgaag tagcggttat ttttttgttt gatttggatt
Pstl
51 ttctaacaag caaagatgag atttccttca atttttactg cagttttatt aagattgttc gtttctactc taaaggaagt taaaaatgac gtcaaaataa » MFalpha > m r f p s i f t a v 1
101 cgcagcatcc tccgcattag ctgctccagt caacactaca acagaagatg gcgtcgtagg aggcgtaatc gacgaggtca gttgtgatgt tgtcttctac > MFalpha > f a a s s a l a a p v n t t t e d
151 aaacggcaca aattccggct gaagctgtca tcggttactc agatttagaa tttgccgtgt ttaaggccga cttcgacagt agccaatgag tctaaatctt > MFalpha > e t a q i p a e a v i g y s d l e
201 ggggatttcg atgttgctgt tttgccattt tccaacagca caaataacgg cccctaaagc tacaacgaca aaacggtaaa aggttgtcgt gtttattgcc > MFalpha > g d f d v a v l p f s n s t n n
251 gttattgttt ataaatacta ctattgccag cattgctgct aaagaagaag caataacaaa tatttatgat gataacggtc gtaacgacga tttcttcttc > MFalpha > g l l f i n t t i a s i a a k e e
Hindi I I -+
301 gggtaagctt ggataaaaga cccattcgaa cctattttct
> MFalpha » g v s l d k r
formed by annealing complementary six single stranded oligonucleotides with the sequences
• 5'TTAAGAGTCCAATTAGCTTCATCGCCAATAAAAAAACAAACTAAACCT AATTCTAACAAGCAAAGATGAGATTTCCTTCAATTTTTACTGCAGTTTTA 3';
• 5' TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTT
AGAAGGGGATTT3' ;
• 5' CGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGGGTTATT GTTTATAAΆTACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTAA GCTTGGATAAAAGA3' ;
• 5' TCTTTTATCCAAGCTTACCCCTTCTTCTTTAGCAGCAATGCTGGCAAT AGTAGTATTTATAAACAATAACCCGTTATTTGTGCTGTTGGAAAATGGCA
AAAC3' ;
• 5'AGCAACATCGAAATCCCCTTCTAAATCTGAGTAACCGATGACAGCTTC
AGCCGGAATTTGTGCCGTTTCATCTTCTGTTGTAGTGTTGACTGGAGCAG CTAATGCGGAGG3' ; and
• 5'ATGCTGCGAATAAAACTGCAGTAAAAATTGAAGGAAATCTCATCTTTG CTTGTTAGAATTAGGTTTAGTTTGTTTTTTTATTGGCGATGAAGCTAATT GGACTC3' . Plasmid mρl9.7 (EP-A-248 637) was digested to completion with XhOi3 phenol/chloroform extracted and ethanol precipitated. The recovered DNA was then blunt ended with the Klenow fragment of E. coli DNA polymerase I to remove the XIwI overhang, phenol/chloroform extracted, and ethanol precipitated. The recovered DNA was digested to completion with Xbal. The digestion products were resolved by agarose gel electrophoresis and the 1.07-kb blunt end- Xbal mpl9.7 fragment recovered using the GeneClean III ldt (Q-bio Gene).
The rHA expression disintegration vector pDB2287 (Figure 25) was created by ligating the Notl expression cassette from pDB2284 into Notl cut pSAC35 (Sleep et al, 1991, Bio/Technology 9, 183-187 and EP 431 880).
The uraS auxotrophic mutant of the AH22 histidine revertant described in Example 4 was co-transformed to both leucine and uracil prototrophy with plasmids pDB2244 and YCplac33, or pDB2244 and pTPC17, or pDB2286 and YCplac33, or pDB2286 and pTPC17, or pDB2287 and YCplac33, or pDB2287 and pTPC17. Transformation was by a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2 (Elble, 1992, op. cit.; Ito et al , 1983, op. cit). Transformants were selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates.
Two transformants for each strain were inoculated into 1OmL BMMD in 5OmL shake flasks and incubated in an orbital shaker at 300C, 200rpm for 4-days. Culture supematants were harvested and the recombinant human albumin (rHA) titres compared by SDS-PAGE (Figure 26 A-C) and densitometric analysis (Figure 26 D). The results are summarised in Table 8, below. Table 8: Increased rHA secretion by overexpression of SILl, LHSl and JEMl (pTPC17) from three distinct leader sequences.
Figure imgf000266_0001
The results indicated that the rHA titres were increased by transformation with pTPC017 relative to the control plasmid YCplac33. Increases in rHA titres varied between the different expression constructs in the range of 14.5 - 29.1% demonstrating the beneficial effect of LHSl, JEMl and SILl on rHA secretion was not restricted to a specific secretory leader sequence. Thus, for example, it is clear that the beneficial effect of LHSl, JEMl and SILl on rHA secretion was not restricted by features of the leader sequence at the amino acid or DNA sequence level, or by configuration (pre or pre-pro) or whether or not the secretory leader sequence contained N-linked glycosylation sites.
EXAMPLE 9
This example describes the increased secretion of recombinant granulocyte macrophage colony stimulating factor (GM-CSF) from a 2-micron based plasmid by over-expression of LHSl, JEMl and SILl from the centromeric vector pTPC17.
A cDNA for human GM-CSF was obtained from plasmid pBBG12 (R&D Systems Europe Ltd.) cloned between the Hindlϊl and EcoRI sites of the pUC18 polylinker. The DNA sequence of the human GM-CSF cDNA (Figure 27) incorporated an N-terminal Met codon. Oligonucleotides SINKl and SINK 2 were synthesised to construct a linker which would reconstruct the JiS AJMFa-I fusion leader as taught in WO 90/01063, coupled to GM-CSF up to the BstElI site.
SINKl: 5'GTACCAAGCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTT ATTCCAGGAGCTTGGATAAAAGAGCACCCGCCCGS'
SINK2: 5' GTGACCGGGCGGGTGCTCTTTTATCCAAGCTCCTGGAATAAGC CGAGCTAAAGAGAAAAAGAAGGGAAATAAAGCTTGS'
A 380bp BsfElVBamRl GMCSF fragment was isolated from pBBG12 and ligated into pUC19 Asp7WBamΗl along with the Asp7WBstEII SINK1/2 linker above, to create pDB2095. Accordingly, the GM-CSF cDNA, linked to the HSA/ 'MFa-I fusion secretion leader, was available on a Hindlll fragment suitable for subcloning into pAYE441 (as described in WO 2004/009819, Example 1 and Figure 5) to create pDB2102 in which the GM-CSF cDNA was now present on a Notl expression cassette, comprising the PRBl promoter, the RSAJMFa-I fusion secretion leader and the ADHl terminator. The GM-CSF Notl expression cassette was isolated and subcloned into pSAC35 (Sleep et al, 1991, Biotechnology (NY), 9, 13 and EP 431 880) linearised with Notl to create plasmid pDB2109 (Figure 28).
The S. cerevisiae Control Strain {uraS), as described above in Example 1, was co- transformed to both leucine and uracil prototrophy with plasmids pDB2109 (Figure 28) and either YCplac33 or pTPC17 (Figure 15). Transformation was by a modified lithium acetate method (Sigma yeast transformation kit, YEAST-I, protocol 2 (Elble, 1992, op. cit; Ito et al, 1983, op. cit). Transformants were selected on BMMD-agar plates, and subsequently patched out on BMMD-agar plates.
Transformants of each strain were inoculated into 1OmL BMMD in 5OmL shake flasks and incubated in an orbital shaker at 3O0C, 200rpm for 4-days. Culture supernatants were harvested and the recombinant GM-CSF titres compared by SDS-PAGE and densitometry analysis (Figure 29 A and B). The results of the densitometric analysis are also provided in Table 9, below.
Table 9: Increased GM-CSF production as determined by SDS-PAGE and densitometric analysis
Figure imgf000268_0001
The results indicated that the recombinant GM-CSF titres in supernatants of BMMD shake flask cultures were greater than 50% higher when pTPC17 was present.

Claims

1. A host cell suitable for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over- expression of two or more helper proteins selected from a DnaJ-like protein (such as JEMl)3 an Hsp70 family protein (such as LHSl) and SILl5 wherein at least one of the over-expressed two or more helper proteins is selected from JEMl, LHSl and SILl, and wherein the DnaJ- like protein is not SCJl .
2. A host cell according to Claim 1 wherein the host cell is genetically modified to cause over-expression of-
(a) a DnaJ-like protein and an Hsp70 family protein; or (b) a DnaJ-like protein and SILl ; or
(c) an Hsp70 family protein and SILl .
3. A host cell suitable for enhanced production of a protein product of choice characterised in that the host cell is genetically modified to cause over- expression of three or more helper proteins, wherein the three or more helper proteins comprise a DnaJ-like protein, an Hsp70 family protein and SILl5 and wherein the DnaJ-like protein is not SCJl.
4. A host cell according to any one of the preceding claims wherein the Hsp70 family protein is a protein that localises to the lumen of the ER.
5. A host cell according to any one of the preceding claims wherein the Hsp70 family protein is not a prokaryotic Hsp70 family protein, and is preferably a eukaryotic (such as a yeast) Hsp70 family protein.
6. A host cell according to any one of the preceding claims wherein the Hsp70 family protein is LHSl5 KAR2, SSAl5 SSA2, SSA3, SSA4, SSEl5
SSE2, SSBl5 SSB2 or ECMlO5 preferably LHSl.
7. A host cell according to any one of the preceding claims wherein the DnaJ- like protein is a protein that localises to the ER membrane.
8. A host cell according to any one of the preceding claims wherein the DnaJ- like protein is not a prokaryotic DnaJ-like protein, and is preferably a eukaryotic (such as a yeast) DnaJ-like protein.
9. A host cell according to any one of the preceding claims wherein the DnaJ- like protein is selected from JEMl, MDJl, MDJ2, SEC63, YDJl, XDJl,
APJl, SISl, DJPl, ZUOl, SWA2, JJJl, JJJ2, JJJ3, CAJl, CWC23, PAM18, JACl, JIDl, SCJl, HLJl and ERJ5, and is preferably JEMl.
10. A host cell according to any one of the preceding claim wherein the host cell is further genetically modified to cause over-expression of at least one, two, three, four, five, six or seven proteins involved in the formation of disulphide bonds in other proteins selected from the group consisting of EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
11. A host cell that is suitable for enhanced production of a protein product of choice characterised in that the host cell comprises a first gene encoding a first helper protein selected from JEMl, LHSl or SILl5 or a variant thereof, and a second gene encoding a desired protein product of choice, wherein the host cell is genetically modified to cause over-expression of the first helper protein, and-
(a) wherein the first and second genes are not both present within the host cell on the same 2μm-family plasmid; and
(b) wherein the host cell is not genetically modified to cause over- expression of a further helper protein that is different from the first helper protein and is selected from the group consisting of AHAl, CCT2, CCT3, CCT4, CCT5, CCT6, CCT7, CCT8, CNSl5 CPR3, CPR6, EROl, EUGl5 FMOl5 HCHl, HSPlO, HSP12, HSP104, HSP26, HSP30, HSP42, HSP60, HSP78, HSP825 JEMl, MDJl, MDJ2, MPDl5 MPD2, PDIl, PFDl, ABCl5 APJl5 ATPI l5 ATP12, BTTl5 CDC37, CPR7, HSC82, KAR2, LHSl5 MGEl5 MRSI l,
NOBl, ECMlO3 SSAl5 SSA2, SSA3, SSA4, SSCl5 SSE2, SILl, SLSl, ORMl5 ORM2, PERl, PTC2, PSEl5 UBI4 and HACl or a truncated intronless HACl.
12. The host cell of Claim 11 wherein the host cell does not comprise a recombinant copy5 such as a plasmid encoded copy, or a chromosomally integrated recombinant copy, of a gene encoding the further helper protein.
13. The host cell of Claim 11 or 12 wherein the first helper protein is the only helper protein that is over-expressed by the host cell.
14. A host cell according to any preceding claim wherein the protein product of choice is a heterologous protein and/or comprises a leader sequence effective to cause secretion, preferably in yeast.
15. A host cell according to any preceding claim wherein the protein product of choice is a eukaryotic protein, or a fragment or variant thereof, preferably a vertebrate or a fungal (such as a yeast) protein.
16. A host cell according to any preceding claim wherein the protein product of choice is a commercially useful protein.
17. A host cell according to any preceding claim wherein the protein product of choice comprises 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 dAb, Fab' fragments, F(ab')2, scAb, scFv or scFv fragment), a Kunitz domain protein (such as aprotinin and amyloid precursor protein), interferons (such as interferon α species and subspecies, interferon β species and sub-species, interferon γ species and subspecies), interleulάns (such as ILlO, ILI l and IL2), leptin, CNTF and fragments thereof (such as CNTFAX1 sXAxokine™)), ILl -receptor antagonist, erythropoietin (EPO) and EPO mimics, thrombopoietin (TPO) and TPO mimics, prosaptide, cyanovirin-N, 5-helix, T20 peptide, T 1249 peptide, HIV gp41, HIV gpl20, uroldnase, prourokinase, tPA, hirudin, platelet derived growth factor, parathyroid hormone, proinsulin, insulin, glucagon, glucagon-lilce 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-tlirombin, von Willebrand's factor, ^ -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, inter- alpha trypsin inhibitor, antithrombin III, apo-lipoprotein species, Protein C, Protein S, or a variant or fragment of any of the above, or a fusion of albumin and any of the above.
18. A host cell according to any preceding claim wherein the protein product of choice comprises the sequence of albumin or a variant or fragment thereof.
19. A host cell according to any preceding claim wherein the protein product of choice comprises the sequence of a transferrin family member, preferably transferrin or lactoferrin, or a variant or fragment thereof.
20. A host cell according to any preceding claim wherein the protein product of choice comprises a fusion protein, such as an albumin or a transferrin family member or a variant or fragment of either, fused directly or indirectly to the sequence of another protein.
21. A host cell according to any preceding claim comprising a polynucleotide sequence that encodes the protein product of choice.
22. A host cell according to Claim 21 wherein the polynucleotide sequence that encodes the protein product of choice is an exogenous polynucleotide.
23. A host cell according to Claim 22 wherein the exogenous polynucleotide is integrated into the chromosome of the host cell.
24. A host cell according to Claim 22 wherein the exogenous polynucleotide is present in the host cell as part of a replicable vector, such as a plasmid.
25. A method for producing a protein product of choice, the method comprising:
(a) providing a host cell as defined by any one of Claims 21 to 24; and
(b) growing the host cell;
thereby to produce a cell culture or recombinant organism comprising an increased level of the protein product of choice compared to the level of production of the protein product of choice achieved by growing, under the same conditions, the same host cell that has not been genetically modified to cause over-expression of one or more helper proteins.
26. The method of Claim 25 wherein the step of growing the host cell involves culturing the host cell in a culture medium.
27. The method of Claim 25 or 26 further comprising the step of purifying the thus expressed protein product of choice from the cultured host cell, recombinant organism or culture medium.
28. The method of Claim 27 further comprising the step of formulating the purified protein product of choice with a carrier or diluent and optionally presenting the thus formulated protein in a unit dosage form.
29. The method of Claim 27 further comprising the step of lyophilising the thus purified protein product of choice.
30. Use of a polynucleotide in the preparation of a host cell as defined by any one of Claims 1 to 24, by transformation of a host cell with the polynucleotide, wherein the polynucleotide comprises a sequence encoding a helper protein selected from the list comprising —
(a) a chaperone selected from a DnaJ-like protein (such as a DnaJ-like protein as defined by any one of Claims 7 to 9), an Hsp70 family protein (such as a Hsp70 family protein as defined by any one of Claims 4 to 6), and SILl, and wherein the DnaJ-like protein is not
SCJl; and
(b) a protein involved in the formation of disulphide bonds in other proteins selected from EROl, ERV2, EUGl, MPDl, MPD2, EPSl and PDIl.
31. Use of a polynucleotide in the preparation of a host cell as defined by any one of Claims 1 to 24, by transformation of a host cell according to any one of Claims 1 to 20 with the polynucleotide, wherein the polynucleotide comprises a sequence encoding a protein product of choice as defined by any one of Claims 14 to 20.
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