WO2002048187A2 - Secretion signal peptides, their dna sequences, expression vectors for eukaryotic cells that can be produced with the same, and use thereof for biotechnological production of proteins - Google Patents

Secretion signal peptides, their dna sequences, expression vectors for eukaryotic cells that can be produced with the same, and use thereof for biotechnological production of proteins Download PDF

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WO2002048187A2
WO2002048187A2 PCT/EP2001/014588 EP0114588W WO0248187A2 WO 2002048187 A2 WO2002048187 A2 WO 2002048187A2 EP 0114588 W EP0114588 W EP 0114588W WO 0248187 A2 WO0248187 A2 WO 0248187A2
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expression
sequence
expression vector
eukaryotic cells
eukaryotic
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German (de)
French (fr)
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WO2002048187A3 (en
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Manfred Schmitt
Uwe WÖLK
Peter Wagner
Tatjana Zagorc
Tanja Heintel
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Phylos, Inc.
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Publication of WO2002048187A3 publication Critical patent/WO2002048187A3/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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence

Definitions

  • Secretion signal peptides their DNA sequences, expression vectors which can be produced therewith for eukaryotic cells and their use for the biotechnological production of proteins
  • the invention relates to peptide secretion signal sequences and nucleic acid sequences coding for them, expression vectors for eukaryotic cells, the cell constructs transfected with these vectors and the use of the vectors and cell constructs for the biotechnological production of proteins.
  • Acetylation which are only carried out in higher organisms.
  • yeasts In addition to systems in mammalian (CHO, BHK, COS, etc.) or insect cells (SF9 cells), yeasts have the advantage as eukaryotic expression systems that they multiply just as quickly as bacteria and can be cultivated inexpensively in the laboratory without great safety measures.
  • a large number of genetic methods for the investigation of molecular biological issues are now also available for yeasts.
  • genes can easily be deleted from a genome, or foreign genes can be introduced into the cells by introducing extrachromosomal plasmids or by integration into the genome. By using different promoters, the expression of these genes is also possible in different strengths or even in a regulatable manner.
  • yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis and for some time now also Schizosaccharomyces pombe have found their way into biotechnological laboratories and production facilities as expression strains (Lu, Y. Bauer, JC, Greener, A. (1997) Gene 200: 135-144).
  • recombinant proteins are not secreted into the extracellular medium but are deposited in the cytoplasm. To isolate the desired protein, the cells must therefore be disrupted and separated from the cell debris thus obtained and the residual proteome of the yeast. However, this represents a large expenditure of time and money. It is therefore desirable to secrete recombinant proteins from a cell in order to simplify production and purification on an industrial scale.
  • Proteins can only be secreted if they contain an N-terminal, hydrophobic secretion and processing sequence that ensures protein import into the lumen of the endoplasmic reticulum (ER), the most important step of the secretion machinery.
  • signal sequences of endogenous proteins such as that of an invertase, an acid phosphatase, or the pheromone factor P have been fused with the protein to be expressed.
  • the present invention is based on the object of providing vectors for the efficient secretory expression of genes in eukaryotic cells and thus an efficient method for the biotechnological production of proteins. It could surprisingly be shown that fusion proteins which have an N-terminal amino acid sequence of the preprotoxin (pptox) of the killer virus K28 persistent in the cytoplasm of S. cerevisiae with the sequence
  • the sequence Seq. Id. No. 1 or a functional variant thereof acts as a secretion signal sequence. It is also advantageous that the secretion signal sequences described have their secretory effect in different eukaryotic host cells, in particular in yeasts. It is particularly surprising that fusion proteins which result from a fusion of the signal sequence Seq. Id. No. 1 of the K28 preprotoxin and a heterologous protein are secreted with a higher yield than the mature toxin in the naturally infected yeast host S. cerevisiae.
  • functional variants in connection with the fusion proteins according to the invention are understood to mean amino acid sequences with a sequence homology of at least 80%, which are suitable as a secretion signal.
  • allelic variants are included in the term functional variant.
  • the fusion proteins can be post-translationally modified, e.g. B. glycosylated, phosphorylated or acetylated.
  • peptides can be used as the secretion signal sequence which have a fragment of at least 20 amino acids according to Seq. Id. No. 1 included.
  • Another object of the present invention are those for a peptide secretion signal according to Seq. Id. No. 1 or one of its functional variants encoding DNA sequence (S / P). DNA sequences according to Seq are preferred. Id. No. 2 or a functional variant of this sequence.
  • a functional variant in connection with the DNA sequence according to the invention means a DNA sequence with a sequence homology of at least 70%, preferably of at least 90%, which codes for a peptide secretion signal.
  • the term functional variant includes, in particular, all allelic sequence variants and all sequences which, under stringent conditions, have the sequence Seq. Id. No. 2 hybridize and their allelic sequence variants. Common hybridization conditions (e.g. 60 ° C, 0.1xSSC, 0.1% SDS) can be used for this.
  • expression vectors which contain a promoter and the S / P secretion signal sequence of the ppfox gene of the virus K28 according to Seq. Id. No. 2 or contain a functional variant of this sequence with a sequence homology of at least 70%, the target sequence in question lying in the 3 ⁇ direction to the promoter and in the open reading frame to the S / P secretion signal sequence.
  • the expression vector preferably contains the sequence regions of the ppfox gene required for splicing the K28 ppfox gene transcript or a functional, ie splicable, variant thereof with a homology of at least 70%.
  • promoters inducible promoters such as. B. the nmtl promoter is used.
  • Proven promoters are e.g. B. the ADH-2 promoter for expression in yeast (Radorel et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1 - 0127839) or the early SV40 promoter or the LTR promoters e.g. B. from MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).
  • yeast restorel et al. (1983), J. Biol. Chem. 258, 2674
  • the baculovirus polyhedrin promoter for expression in insect cells see, for example, EP-B1 - 0127839
  • the early SV40 promoter or the LTR promoters e.g. B. from MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).
  • the expression vectors according to the invention can contain further functional sequence regions, such as, for. B. a replication starting point, operators, termination signals such. B. contain the nm - ⁇ - termination signal, or selection markers, repressors, activators coding sequences.
  • yeast z. B the pREP-K28 vector, the pINT-K28 vector, the pTZ ⁇ / ⁇ vector or the pTZsp vector (FIGS. 2 and 5), which are based on freely available vectors, into which an S / P signal sequence is shown of the K28 virus was cloned.
  • eukaryotic expression vectors which are suitable for expression in Saccharomyces cerevisiae are e.g. Vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767), for expression in insect cells e.g. Baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells e.g. B. SV40 vectors suitable, which are commonly available.
  • Heterologous or homologous genes which are to be expressed in eukaryotic cells can be cloned into the vectors according to the invention. These genes can either lie directly in the open reading frame behind the S / P signal sequence of the K28 virus or can be introduced into the ⁇ or ⁇ subunit of the K28 ppfox gene, so that the target gene product with the S / P signal sequence is more post-translational Level to a sequence encoding a fusion protein can be processed.
  • interesting target genes are primarily eukaryotic genes, such as. B. for eukaryotic structural proteins, enzymes, receptors, repressors, transcription factors or ion channels. However, the expression of artificial, artificially modified or mutated coding nucleic acid sequences is also conceivable.
  • the expression vectors according to the invention open up by the choice of a homologous eukaryotic host cell for the expression of certain proteins, to produce proteins with their native post-translational modification pattern more simply and more efficiently.
  • the expression of target genes using known host cells such as e.g. B. S. cerevisiae, can be further improved.
  • a preferred host organism is the yeast S. pombe, which has hitherto hardly been used as an expression organism. S. pombe is closer to the higher eukaryotes than z. B. S. cerevisiae, in addition, very high yields of secreted protein based on cell density are obtained with S. pombe.
  • the present invention further provides expression systems from eukaryotic host cells which are transfected with the eukaryotic expression vectors described above.
  • Yeasts in particular S. pombe, are preferred as host cells.
  • other proven yeasts such as. B. the genera Aspergillus, Schwanniomyces, Kluyveromyces, Yarrowia, Arxula, Saccharomyces, Schizosaccharomyces, Hansenula, Pichia, Hanseniaspora, Zygosaccharomyces, Ustilago, Debaryomyces, Cryptococcus, Rhodotorula, Trichosporon, Kluyveromyces, Toruy.
  • the present invention furthermore relates to the use of the vector systems according to the invention for cloning target genes and for Transfection of eukaryotic cells and the use of expression systems generated in this way for the cultivation and production of proteins.
  • additives such as. B. to transfer protease inhibitors.
  • FIGS 1 to 7 serve to clarify the invention and are briefly explained below.
  • Fig. 1 Processing of the K28 preprotoxin in yeast.
  • the schematic structure of the unprocessed toxin precursor is shown. Furthermore, the cleavage sites for a signal peptidase (S / P) and the processing sites of the Kex2p, Krpl p and Kex1 p proteases are marked. Potential N-glycosylation sites and a disulfide bridge between Cys 56 ( ⁇ subunit) and Cys 340 ( ⁇ subunit) are marked with -CHO or -SS-.
  • the K28 toxin precursor consists of an N-terminal signal sequence (S / P), followed by a hydrophobic ⁇ subunit, which in turn is separated from a more hydrophilic ⁇ subunit via the N-glycosylated y subunit.
  • S / P N-terminal signal sequence
  • hydrophobic ⁇ subunit hydrophobic ⁇ subunit
  • hydrophilic ⁇ subunit hydrophilic ⁇ subunit
  • the signal sequence is removed by a signal peptidase.
  • the resulting protoxin is further processed by the Kex1 p / Kex2p endoproteases found in the Golgi apparatus, so that finally an active toxin consisting of an ⁇ and ⁇ subunit, which are connected by a disulfide bridge, is secreted.
  • the area of the gap yeast nm - ⁇ - promoter for the transcription initiation is with
  • Lines represent sequences from yeast, thin lines represent Escherichia coli pUC19 sequences (oriE, E. coli origin of replication; AmpR, ⁇ -lactamase Gene; arsl, autonomous replicating sequence from S. pombe; LEU2, S. cerevisiae LEU2 gene; S. pombe ura4 + and Ie ⁇ 1 * genes).
  • Fig. 3 Thiamine-regulated toxin expression of the recombinant S. pombe strains. The filter of a Western blot is shown, on which the active toxin is detected with a polyclonal antiserum against the ⁇ -subunit. Lanes 1 and 3, culture supernatants of repressed S. pombe (pREP-K28 and plNT-K28) cultures; Lanes 2 and 4, culture supernatants of induced S. pombe (pREP-K28 and plNT-K28) cultures; Lanes R and I (negative control), supernatants of two S.
  • pombe transformants which carry either only the pREP1 or the plNT5 plasmid; Lane C positive control, partially purified mature K28 toxin; Lane S, pre-stained protein fraction for molecular weight determination.
  • the large arrow marks the 21 kDa active, heterodimeric toxin; the two small arrows mark the tetramer derivative ( ⁇ / ß) 2 which forms spontaneously in an SDS-PAGE under non-reducing conditions or the monomeric ß subunit.
  • Fig. 4 Comparison of the recombinant or homologous toxin secretion in S. pombe or S. cerevisiae.
  • the filter of a Western blot is shown, on which the active toxin is detected with a polyclonal antiserum against the ⁇ -subunit (FIG. 4a).
  • the application scheme is as follows: lanes 1 and 2, extracellular samples each of an induced culture of the S. pombe strain, which secretes the toxin from the episomally present plasmid (pREP-K28) or the chromosomally integrated plasmid (plNT-K28); Lane 3 extracellular samples of the S. cerevisiae strain, which expresses the K28-ppfox gene product episomally (pFR5-TPI) or the virus-infected S.
  • the area of the gap yeast ⁇ m ⁇ promoter for the transcription initiation is marked with Pnmtl and that for the transcription termination with Tnmtl.
  • S / P symbolizes the processing and secretion sequence of the K28 killer toxin, GFP the green fluorescent protein from Aequorea victoria.
  • Bold lines represent sequences from yeast, thin lines represent Escherichia coli pUC19 sequences (oriE, E. coli origin of replication; AmpR, ⁇ -lactamase gene; arsl, autonomous replicating sequence from S. pombe; .URA4, S. cerevisiae).
  • Fig. 6/7 Secretion of heterologous fusion proteins in S. pombe. Filters from Western blots are shown, on which the secretion of the respective fusions with a specific antibody directed against the GFP protein is detected. The assignment of the two filters is the same, with FIG. 6 showing the GFP secretion from plasmid pTZ ⁇ / ⁇ and FIG. 7 showing GFP secretion from plasmid pTZsp.
  • Lanes 1 and 3 Culture supernatants of repressed S. pomi e cultures which either carry only the plasmid alone or the expression plasmid for secretion of the GFP as a negative control.
  • Lanes 2 and 4 Culture supernatants of induced S. pombe cultures which either carry only the plasmid alone or the expression plasmid for secretion of the GFP as a negative control;
  • Lane S pre-stained protein fraction for molecular weight determination;
  • Lane C positive control, purified recombinant GFP protein.
  • the plasmids for the episomal or chromosomal-integrated expression of the K28 killer toxin are shown schematically in FIG. 2.
  • a 1048 bp Xhol / BglII fragment of the K28 killer toxin-encoding yeast plasmid was used for generation PPGK-M28-1 (Schmitt, MJ and Tipper, DJ. (1995) Virology 213: 341-351) was cloned into the expression vectors pREP1 and pINT5 from S. pombe restricted and linearized with Sall / BamHI.
  • the proteins to be expressed can be controlled via the thiamine-regulable promoter nmtl.
  • the vectors were then transformed into the two strains of the host yeast S. pombe using the lithium acetate method.
  • the transformants were selected on minimal medium without uracil (for plNT-K28) or without leucine (for pREP-K28).
  • PINT-K28 and pREP-K28 positive yeast transformants were tested for their killer phenotype in a standard test, the agar diffusion assay, on methylene blue stained agar plates with the K28 toxin-sensitive S. cerevisae strain 192.2d (data not shown).
  • 600 ⁇ l of cell culture supernatant from the S. pombe strain with the episomal plasmid (pREP-K28) or the S. pombe strain with the chromosomal plasmid (plNT-K28) at a density of 5 ⁇ 10 7 cells per ml were used repressing (thiamine-containing, 25 ⁇ M) or inducing (thiamine-free) medium removed, ethanol precipitated and separated under non-reducing conditions on a 10-22.5% gradient gel (SDS-PAGE).
  • the active toxin was detected by a polyclonal rabbit antiserum (primary antibody) against the ⁇ -subunit of the active toxin (secondary antibody: goat anti rabbit IgG alkaline phosphatase). After incubation of the membrane with an NBT / BCIP staining solution, the ß-subunit of the toxin could be made visible through a staining reaction.
  • FIG. 5 The constructs for the episomal secretion of the green fluorescent protein (GFP) are shown schematically in FIG. 5.
  • a DNA which was encoded for the GFP protein from Aequorea victoria was cloned as a BamHI / BamHI fragment with a Bglll / Bglll fragment of the expression vector pTZ ⁇ / ⁇ or with a Bglll / Bglll fragment of the expression vector pTZsp.
  • hybrid plasmids pTZ ⁇ / ß and pTZsp resulted from sequences of the expression vector pREP4x and the K28 ppfox gene or the expression vector pREP4x and the signal sequence S / P of the ppfox gene (Basi, G., Schmid, E., Maundrell, K. (1993) Gene 123: 131-136; Schmitt, MJ and Tipper, DJ. (1995) Virology 213: 341-351).
  • a fusion protein consisting of the ⁇ and the ⁇ subunit of the active toxin and the GFP protein (pTZ / ⁇ -GFP)
  • a fusion consisting of the signal sequence (S / P) and the GFP protein expressed pTZsp-GFP
  • S / P signal sequence
  • pTZsp-GFP GFP protein expressed
  • the recombinant strains were first cultured for 24 hours in uracil-containing EEM medium and then for repression in the same medium for 24 hours in the presence of 25 ⁇ M thiamine (Fig. 6/7). to subsequent depression and thus expression of the fusion proteins were the
  • Antibody response can be detected (specific primary antibody: anti GFP
  • Mouse IgG Secondary antibody. anti-mouse IgG peroxidase).
  • the resulting chemiluminescence could be visualized by exposure to an X-ray film using the Röche Diagnostics substrate.

Abstract

The invention relates to peptidic secretion signal sequences and nucleic acids coding for the same, to expression vectors for eukaryotic cells, to cell constructs which are transfected with these vectors and to the use of said vectors and cell constructs for the efficient biotechnological production of proteins with eukaryotic cells.

Description

Sekretionssignalpeptide, deren DNA-Sequenzen, damit herstellbare Expressionsvektoren für eukaryotische Zellen und deren Verwendung zur biotechnologischen Herstellung von Proteinen Secretion signal peptides, their DNA sequences, expression vectors which can be produced therewith for eukaryotic cells and their use for the biotechnological production of proteins
Beschreibungdescription
Die Erfindung betrifft peptidische Sekretionssignalsequenzen sowie für diese kodierende Nukleinsäuresequenzen, Expressionsvektoren für eukaryotische Zellen, die mit diesen Vektoren transfizierten Zellkonstrukte sowie die Nutzung der Vektoren und Zellkonstrukte zur biotechnologischen Herstellung von Proteinen.The invention relates to peptide secretion signal sequences and nucleic acid sequences coding for them, expression vectors for eukaryotic cells, the cell constructs transfected with these vectors and the use of the vectors and cell constructs for the biotechnological production of proteins.
Traditionell stehen für die Expression und Reinigung von Proteinen bakterielleBacterial are traditionally used for the expression and purification of proteins
Systeme wie die aus Escherichia coli oder Bacillus subtilis zur Verfügung. Sind die zu exprimierenden Proteine eukaryotischen Ursprungs, kann die Expression in einem prokaryotischen Wirt aber problematisch sein. Die Aktivität und Produktionsmenge vieler Proteine hängt zu einem erheblichen Teil von posttranslationalen Modifikationen, wie N-Glykosylierung, Phosphorylierung, oderSystems such as those from Escherichia coli or Bacillus subtilis are available. However, if the proteins to be expressed are of eukaryotic origin, expression in a prokaryotic host can be problematic. The activity and production amount of many proteins depends to a large extent on post-translational modifications such as N-glycosylation, phosphorylation, or
Acetylierung ab, die nur in höheren Organismen durchgeführt werden.Acetylation, which are only carried out in higher organisms.
Neben Systemen in Säugetier- (CHO, BHK, COS etc.) oder Insektenzellen (SF9- Zellen) haben Hefen als eukaryotische Expressionssysteme den Vorteil, dass sie sich ähnlich schnell wie Bakterien vermehren und sich im Labor kostengünstig ohne große Sicherheitsvorkehrungen kultivieren lassen. Auch stehen für Hefen mittlerweile eine Vielzahl genetischer Methoden zur Untersuchung von molekularbiologischen Fragestellungen zur Verfügung. So können Gene aus einem Genom leicht deletiert, oder fremde Gene durch Einschleusen extrachromosomaler Plasmide oder durch Integration in das Genom zusätzlich in die Zellen eingebracht werden. Durch die Verwendung verschiedener Promotoren wird die Expression dieser Gene auch in unterschiedlicher Stärke oder sogar regulierbar möglich. Vor allem Hefen wie Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis und seit einiger Zeit auch Schizosaccharomyces pombe haben deshalb als Expressionsstämme Einzug in biotechnologische Labors bzw. Produktionsanlagen gehalten (Lu, Y. Bauer, J.C., Greener, A. (1997) Gene 200: 135-144).In addition to systems in mammalian (CHO, BHK, COS, etc.) or insect cells (SF9 cells), yeasts have the advantage as eukaryotic expression systems that they multiply just as quickly as bacteria and can be cultivated inexpensively in the laboratory without great safety measures. A large number of genetic methods for the investigation of molecular biological issues are now also available for yeasts. For example, genes can easily be deleted from a genome, or foreign genes can be introduced into the cells by introducing extrachromosomal plasmids or by integration into the genome. By using different promoters, the expression of these genes is also possible in different strengths or even in a regulatable manner. In particular, yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Kluyveromyces lactis and for some time now also Schizosaccharomyces pombe have found their way into biotechnological laboratories and production facilities as expression strains (Lu, Y. Bauer, JC, Greener, A. (1997) Gene 200: 135-144).
Im allgemeinen werden rekombinante Proteine nicht in das extrazelluläre Medium sezerniert sondern im Zytoplasma abgelagert. Zur Isolierung des gewünschten Proteins müssen die Zellen deshalb aufgeschlossen und von den so erhaltenen Zelltrümmern sowie dem Restproteom der Hefe abgetrennt werden. Dies stellt aber zeitlich und finanziell einen grossen Aufwand dar. Es ist also wünschenswert rekombinante Proteine aus einer Zelle zu sezemieren um die Produktion und Reinigung im großtechnischen Maßstab zu vereinfachen.In general, recombinant proteins are not secreted into the extracellular medium but are deposited in the cytoplasm. To isolate the desired protein, the cells must therefore be disrupted and separated from the cell debris thus obtained and the residual proteome of the yeast. However, this represents a large expenditure of time and money. It is therefore desirable to secrete recombinant proteins from a cell in order to simplify production and purification on an industrial scale.
Proteine können nur sezerniert werden, wenn sie eine N-terminale, hydrophobe Sekretions- und Prozessierungssequenz beinhalten, die den Proteinimport in das Lumen des endoplasmatischen Retikulums (ER), der wichtigste Schritt der Sekretionsmaschinerie, sicherstellt. Hierfür werden bisher Signalsequenzen endogener Proteine, wie die einer Invertase, einer sauren Phosphatase, oder des Pheromons Faktor P mit dem zu exprimierenden Protein fusioniert. Auf diesem Weg konnten aber nur wenige medizinisch und pharmakologisch interessante Proteine in Hefen, wie σ-Amylase der Maus, Antithrombin III des Menschen oder die humane Alkalische Phosphatase, in größeren Mengen aktiv in biologischer Form hergestellt werden (Broker, M., Ragg , H., Karges, H.E. (1987) Biochemical Biophysical Acta 908: 203-213; Sambamurti, K. (1997) In: Foreign gene expression in fission Yeast: S. pombe, Giga-Hama, Y. und Kumagai, H. (eds.) Springer Verlag: 149-158; Tokunaga, M., Kawamura, A., Yonekyu, S. (1993) Yeast 9: 379-387).Proteins can only be secreted if they contain an N-terminal, hydrophobic secretion and processing sequence that ensures protein import into the lumen of the endoplasmic reticulum (ER), the most important step of the secretion machinery. To this end, signal sequences of endogenous proteins, such as that of an invertase, an acid phosphatase, or the pheromone factor P have been fused with the protein to be expressed. In this way, however, only a few medically and pharmacologically interesting proteins in yeast, such as mouse σ-amylase, human antithrombin III or human alkaline phosphatase, could be actively produced in large quantities in biological form (Broker, M., Ragg, H ., Karges, HE (1987) Biochemical Biophysical Acta 908: 203-213; Sambamurti, K. (1997) In: Foreign gene expression in fission Yeast: S. pombe, Giga-Hama, Y. and Kumagai, H. (eds .) Springer Verlag: 149-158; Tokunaga, M., Kawamura, A., Yonekyu, S. (1993) Yeast 9: 379-387).
Ausgehend vom Stand der Technik liegt der vorliegenden Erfindung die Aufgabe zugrunde, Vektoren für die effiziente sekretorische Expression von Genen in eukaryotischen Zellen und somit ein effizientes Verfahren zur biotechnologischen Herstellung von Proteinen zur Verfügung zu stellen. Es konnte überraschend gezeigt werden, dass Fusionsproteine, die eine N-terminale Aminosäuresequenz des Präprotoxins (pptox) des im Zytoplasma von S. cerevisiae persistierenden Killervirus K28 mit der SequenzStarting from the prior art, the present invention is based on the object of providing vectors for the efficient secretory expression of genes in eukaryotic cells and thus an efficient method for the biotechnological production of proteins. It could surprisingly be shown that fusion proteins which have an N-terminal amino acid sequence of the preprotoxin (pptox) of the killer virus K28 persistent in the cytoplasm of S. cerevisiae with the sequence
M E S V S S L F N I F S T I M V N Y K S L V L A L L S V S N L K Y A R GM E S V S S L F N I F S T I M V N Y K S L V L A L L S V S N L K Y A R G
(Seq.ld.No.1)(Seq.ld.No.1)
oder einer funktionellen Variante davon mit einer Homologie oder Identität von mindestens 80%, bevorzugt von mindestens 90%, enthalten, effizient unter Verwendung eukaryotischer Wirtszellen exprimiert und sezerniert werden können, wobei die Sequenz Seq. Id. No. 1 oder eine funktioneile Variante davon als Sekretionssignalsequenz wirkt. Weiterhin vorteilhaft ist, dass die beschriebenen Sekretionssignalsequenzen in unterschiedlichen eukaryontischen Wirtszellen, insbesondere in Hefen, ihre sekretorische Wirkung entfalten. Besonders überraschend ist, dass Fusionsproteine, die aus einer Fusion der Signalsequenz Seq. Id. No. 1 des K28 Präprotoxins und einem heterologen Protein bestehen, mit einer höheren Ausbeute sekretiert werden als das reife Toxin im natürlich infizierten Hefewirt S. cerevisiae.or a functional variant thereof with a homology or identity of at least 80%, preferably of at least 90%, can be efficiently expressed and secreted using eukaryotic host cells, the sequence Seq. Id. No. 1 or a functional variant thereof acts as a secretion signal sequence. It is also advantageous that the secretion signal sequences described have their secretory effect in different eukaryotic host cells, in particular in yeasts. It is particularly surprising that fusion proteins which result from a fusion of the signal sequence Seq. Id. No. 1 of the K28 preprotoxin and a heterologous protein are secreted with a higher yield than the mature toxin in the naturally infected yeast host S. cerevisiae.
Unter funktionellen Varianten im Zusammenhang mit den erfindungsgemäßen Fusionsproteinen werden im Sinne dieser Erfindung Aminosäuresequenzen mit einer Sequenzhomologie von mindestens 80% verstanden, die als Sekretionssignal geeignet sind. Insbesondere allele Varianten sind von dem Begriff der funktionellen Variante miteingeschlossen.For the purposes of this invention, functional variants in connection with the fusion proteins according to the invention are understood to mean amino acid sequences with a sequence homology of at least 80%, which are suitable as a secretion signal. In particular, allelic variants are included in the term functional variant.
Weiterhin können die Fusionsproteine posttranslational modifiziert, z. B. glykosyliert, phosphoryliert oder acetyliert, sein.Furthermore, the fusion proteins can be post-translationally modified, e.g. B. glycosylated, phosphorylated or acetylated.
Weiterhin können als Sekretionssignalsequenz Peptide benutzt werden die ein mindestens 20 Aminosäuren langes Fragment gemäß Seq. Id. No. 1 enthalten.Furthermore, peptides can be used as the secretion signal sequence which have a fragment of at least 20 amino acids according to Seq. Id. No. 1 included.
Ein weiterer Gegenstand der vorliegenden Erfindung sind die für ein peptidisches Sekretionssignal gemäß Seq. Id. No. 1 oder einer seiner funktionellen Varianten kodierende DNA-Sequenz (S/P). Bevorzugt sind DNA-Sequenzen gemäß Seq. Id. No. 2 oder einer funktionellen Variante dieser Sequenz.Another object of the present invention are those for a peptide secretion signal according to Seq. Id. No. 1 or one of its functional variants encoding DNA sequence (S / P). DNA sequences according to Seq are preferred. Id. No. 2 or a functional variant of this sequence.
ATG GAG AGC GTT TCC TCA TTA TTT AAC ATT TTT TCA ACA ATC ATG GTT AAC TAT AAA TCG TTA GTT CTA GCA CTA TTA AGT GTT TCA AAT CTC AAA TAT GCA CGG GGTATG GAG AGC GTT TCC TCA TTA TTT AAC ATT TTT TCA ACA ATC ATG GTT AAC TAT AAA TCG TTA GTT CTA GCA CTA TTA AGT GTT TCA AAT CTC AAA TAT GCA CGG GGT
(Seq. Id. No.2)(Seq. Id. No.2)
Unter einer funktionellen Variante im Zusammenhang mit der erfindungsgemäßen DNA-Sequenz wird im Sinne dieser Erfindung eine DNA-Sequenz mit einer Sequenzhomologie von mindestens 70%, bevorzugt von mindestens 90%, verstanden, die für ein peptidisches Sekretionssignal kodiert. Unter den Begriff der funktionellen Variante fallen insbesondere alle allelen Sequenzvarianten sowie alle Sequenzen, die unter stringenten Bedingungen mit der Sequenz Seq. Id. No. 2 hybridisieren und deren allele Sequenzvarianten. Dazu können gängige Hybridisierungsbedingungen (z. B. 60°C, 0,1xSSC, 0.1% SDS) genutzt werden.For the purposes of this invention, a functional variant in connection with the DNA sequence according to the invention means a DNA sequence with a sequence homology of at least 70%, preferably of at least 90%, which codes for a peptide secretion signal. The term functional variant includes, in particular, all allelic sequence variants and all sequences which, under stringent conditions, have the sequence Seq. Id. No. 2 hybridize and their allelic sequence variants. Common hybridization conditions (e.g. 60 ° C, 0.1xSSC, 0.1% SDS) can be used for this.
Zur sekretorischen Expression von interessanten Genen (Zielgenen), werden Expressionsvektoren genutzt, die einen Promotor und die S/P- Sekretionssignalsequenz des ppfox-Gens des Virus K28 gemäß Seq. Id. No. 2 oder eine funktionelle Variante dieser Sequenz mit einer Sequenzhomologie von mindestens 70% enthalten, wobei die betreffende Zielsequenz in 3Λ-Richtung zum Promotor und im offenen Leseraster zur S/P-Sekretionssignalsequenz liegt.For the secretory expression of interesting genes (target genes), expression vectors are used which contain a promoter and the S / P secretion signal sequence of the ppfox gene of the virus K28 according to Seq. Id. No. 2 or contain a functional variant of this sequence with a sequence homology of at least 70%, the target sequence in question lying in the 3 Λ direction to the promoter and in the open reading frame to the S / P secretion signal sequence.
Die Expressionsvektoren können neben der Sekretionssignalsequenz Seq. Id. No. 2 zusätzlich die a- und/oder ß- und/oder ^-Untereinheit kodierenden Teilbereiche des K28-ppfox-Gens oder Teile davon enthalten. Von besonderem Interesse sind hier die Teilebereiche die proteolytische Spaltsignale enthalten. Bevorzugt enthält der Expressionsvektor die zum Spleißen des K28-ppfox-Gentranskripts nötigen Sequenzbereiche des ppfox-Gens oder eine funktionelle, d. h. spleißbare, Variante davon mit einer Homologie von mindestens 70%. Als Promotoren werden bevorzugt induzierbare Promotoren, wie z. B. der nmtl - Promotor benutzt. Aber es können darüber hinaus alle dem Fachmann bekannten Promotoren, bevorzugt Hefepromotoren genutzt werden. Bewährte Promotoren sind z. B. der ADH-2-Promotor für die Expression in Hefen (Rüssel et al. (1983), J. Biol. Chem. 258, 2674), der Baculovirus-Polyhedrin-Promotor für die Expression in Insektenzellen (s. z.B. EP-B1 -0127839) oder der frühe SV40-Promotor oder die LTR-Promotoren z. B. von MMTV (Mouse Mammary Tumour Virus; Lee et al. (1981) Nature, 214, 228).In addition to the secretion signal sequence Seq. Id. No. 2 additionally contain the partial regions of the K28-ppfox gene encoding the a and / or β and / or ^ subunit or parts thereof. Of particular interest here are the parts areas that contain proteolytic cleavage signals. The expression vector preferably contains the sequence regions of the ppfox gene required for splicing the K28 ppfox gene transcript or a functional, ie splicable, variant thereof with a homology of at least 70%. As promoters inducible promoters such as. B. the nmtl promoter is used. However, it is also possible to use all promoters known to the person skilled in the art, preferably yeast promoters. Proven promoters are e.g. B. the ADH-2 promoter for expression in yeast (Rüssel et al. (1983), J. Biol. Chem. 258, 2674), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1 - 0127839) or the early SV40 promoter or the LTR promoters e.g. B. from MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) Nature, 214, 228).
Die erfindungsgemäßen Expressionsvektoren können weitere funktionale Sequenzbereiche, wie z. B. einen Replikationsstartpunkt, Operatoren, Terminationssignale, wie z. B. das nm-^-Terminationssignal, oder Selektionsmarker, Repressoren, Aktivatoren kodierende Sequenzen enthalten.The expression vectors according to the invention can contain further functional sequence regions, such as, for. B. a replication starting point, operators, termination signals such. B. contain the nm - ^ - termination signal, or selection markers, repressors, activators coding sequences.
Als geeignete Expressionsvektoren für Hefen haben sich z. B. der pREP-K28- Vektor, der pINT-K28-Vektor der pTZα/ß-Vektor oder der pTZsp-Vektor erwiesen (Fig. 2 und 5), die auf frei erhältlichen Vektoren basieren, in die eine S/P- Signalsequenz des K28-Virus kloniert wurde. Weitere Beispiele für verwendbare eukaryotische Expressionsvektoren die sich für die Expression in Saccharomyces cerevisiae eignen sind z. B. die Vektoren p426Met25 oder p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767), für die Expression in Insektenzellen z.B. Baculovirus-Vektoren wie in EP-B1 -0127839 oder EP-B1 -0549721 offenbart, und für die Expression in Säugerzellen sind z. B. SV40-Vektoren geeignet, welche allgemein erhältlich sind.As suitable expression vectors for yeast z. B. the pREP-K28 vector, the pINT-K28 vector, the pTZα / β vector or the pTZsp vector (FIGS. 2 and 5), which are based on freely available vectors, into which an S / P signal sequence is shown of the K28 virus was cloned. Further examples of usable eukaryotic expression vectors which are suitable for expression in Saccharomyces cerevisiae are e.g. Vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767), for expression in insect cells e.g. Baculovirus vectors as disclosed in EP-B1-0127839 or EP-B1-0549721, and for expression in mammalian cells e.g. B. SV40 vectors suitable, which are commonly available.
In die erfindungsgemäßen Vektoren können heterologe oder homologe Gene kloniert werden, die in eukaryotischen Zellen exprimiert werden sollen (Zielgene). Diese Gene können entweder direkt im offenen Leseraster hinter der S/P- Signalsequenz des K28 Virus liegen oder in die α- oder ß-Untereinheit des K28- ppfox-Gens eingebracht sein, so dass das Zielgenprodukt mit der S/P- Signalsequenz auf posttranslationaler Ebene zu einer ein Fusionsprotein kodierenden Sequenz prozessiert werden kann. Interessante Zielgene sind vor allem eukaryotische Gene, wie z. B. für eukaryotische Strukturproteine Enzyme, Rezeptoren, Repressoren, Transkriptionsfaktoren oder lonenkanäle. Aber auch die Expression von künstlichen, künstlich veränderten oder mutierten kodierenden Nukleinsäuresequnezen ist denkbar.Heterologous or homologous genes which are to be expressed in eukaryotic cells (target genes) can be cloned into the vectors according to the invention. These genes can either lie directly in the open reading frame behind the S / P signal sequence of the K28 virus or can be introduced into the α or β subunit of the K28 ppfox gene, so that the target gene product with the S / P signal sequence is more post-translational Level to a sequence encoding a fusion protein can be processed. Interesting target genes are primarily eukaryotic genes, such as. B. for eukaryotic structural proteins, enzymes, receptors, repressors, transcription factors or ion channels. However, the expression of artificial, artificially modified or mutated coding nucleic acid sequences is also conceivable.
Zum einen eröffnen die erfindungsgemäßen Expressionsvektoren durch die Wahl einer homologen eukaryotischen Wirtszelle für die Expression bestimmter Proteine, Proteine mit ihrem nativen posttranslationalen Modifikationsmuster einfacher und effizienter herzustellen, zum anderen kann die Expression von Zielgenen unter Verwendung bereits bekannter Wirtszellen, wie z. B. S. cerevisiae , weiter verbessert werden. Ein bevorzugter Wirtsorganismus stellt die bisher kaum als Expressionsorganismus genutzte Hefe S. pombe dar. S. pombe steht den höheren Eukaryoten näher als z. B. S. cerevisiae, darüber hinaus werden mit S. pombe sehr hohe Ausbeuten an sezerniertem Protein bezogen auf die Zelldichte erhalten.On the one hand, the expression vectors according to the invention open up by the choice of a homologous eukaryotic host cell for the expression of certain proteins, to produce proteins with their native post-translational modification pattern more simply and more efficiently. On the other hand, the expression of target genes using known host cells such as e.g. B. S. cerevisiae, can be further improved. A preferred host organism is the yeast S. pombe, which has hitherto hardly been used as an expression organism. S. pombe is closer to the higher eukaryotes than z. B. S. cerevisiae, in addition, very high yields of secreted protein based on cell density are obtained with S. pombe.
Folglich sind ein weiterer Gegenstand der vorliegenden Erfindung Expressionssysteme aus eukaryotischen Wirtszellen, die mit den oben beschrieben eukaryotischen Expressionsvektoren transfiziert sind. Als Wirtszellen sind Hefen, insbesondere S. pombe bevorzugt. Aber auch die Verwendung anderer bewährter Hefen, wie z. B. die Gattungen Aspergillus, Schwanniomyces, Kluyveromyces, Yarrowia, Arxula, Saccharomyces, Schizosaccharomyces, Hansenula, Pichia, Hanseniaspora, Zygosaccharomyces, Ustilago, Debaryomyces, Cryptococcus, Rhodotorula, Trichosporon, Kluyveromyces, Torulopsis oder Williopsis ist möglich.Accordingly, the present invention further provides expression systems from eukaryotic host cells which are transfected with the eukaryotic expression vectors described above. Yeasts, in particular S. pombe, are preferred as host cells. But also the use of other proven yeasts, such as. B. the genera Aspergillus, Schwanniomyces, Kluyveromyces, Yarrowia, Arxula, Saccharomyces, Schizosaccharomyces, Hansenula, Pichia, Hanseniaspora, Zygosaccharomyces, Ustilago, Debaryomyces, Cryptococcus, Rhodotorula, Trichosporon, Kluyveromyces, Toruy.
Verfahren zur Transfektion und Kultivierung der Wirtszellen sind dem Fachmann bekannt. Im Vergleich zur zytoplasmatischen Expression rekombinanter Proteine ermöglicht die hier beschriebene Sekretion eine schnelle und kostengünstige Produktion interessanter Proteine, z. B. von medizinisch-pharmakologisch bedeutenden Proteinen.Methods for transfection and cultivation of the host cells are known to the person skilled in the art. In comparison to the cytoplasmic expression of recombinant proteins, the secretion described here enables fast and inexpensive production of interesting proteins, e.g. B. of medico-pharmacologically important proteins.
Ein weiterer Gegenstand der vorliegenden Erfindung ist die Verwendung der erfindungsgemäßen Vektorsysteme zur Klonierung von Zielgenen und zur Transfektion von eukaryotischen Zellen sowie die Verwendung so erzeugter Expressionssysteme zur Kultivierung und Herstellung von Proteinen.The present invention furthermore relates to the use of the vector systems according to the invention for cloning target genes and for Transfection of eukaryotic cells and the use of expression systems generated in this way for the cultivation and production of proteins.
Bei der sekretorischen Expression von Proteinen kann es vorteilhaft sein, das Expressionsmedium mit Zusatzstoffen, wie z. B. Proteaseinhibitoren zu versetzen.In the secretory expression of proteins, it may be advantageous to add additives such as. B. to transfer protease inhibitors.
Zur Verdeutlichung der Erfindung dienen die Figuren 1 bis 7, die im folgenden kurz erläutert werden.Figures 1 to 7 serve to clarify the invention and are briefly explained below.
Fig. 1 : Prozessierung des K28-Präprotoxins in Hefe.Fig. 1: Processing of the K28 preprotoxin in yeast.
Gezeigt ist die schematische Struktur des unprozessierten Toxin-Vorläufers. Des weiteren sind die Spaltstellen für eine Signal-Peptidase (S/P), und die Prozessierungstellen der Kex2p-, der Krpl p- und der Kex1 p-Proteasen markiert. Potentielle N-Glykosylierungsstellen und eine Disulfidbrücke zwischen Cys56 (α- Untereinheit) und Cys340 (ß-Untereinheit) sind mit -CHO bzw. -S-S- gekennzeichnet.The schematic structure of the unprocessed toxin precursor is shown. Furthermore, the cleavage sites for a signal peptidase (S / P) and the processing sites of the Kex2p, Krpl p and Kex1 p proteases are marked. Potential N-glycosylation sites and a disulfide bridge between Cys 56 (α subunit) and Cys 340 (β subunit) are marked with -CHO or -SS-.
Der K28 Toxin-Vorläufer besteht aus einer N-terminalen Signalsequenz (S/P), gefolgt von einer hydrophoben α-Untereinheit, die wiederum von einer eher hydrophilen ^-Untereinheit über die N-glykosylierte y-Untereinheit getrennt ist. Während der Passage durch den Sekretionsweg wird die Signalsequenz durch eine Signal-Peptidase entfernt. Das daraus entstehende Protoxin wird weiterhin durch die im Golgi-Apparat vorkommenden Kex1 p/ Kex2p Endoproteasen prozessiert, so dass schließlich ein aktives Toxin, bestehend aus einer α- und ß-Untereinheit, die durch eine Disulfidbrücke verbunden sind, sekretiert wird.The K28 toxin precursor consists of an N-terminal signal sequence (S / P), followed by a hydrophobic α subunit, which in turn is separated from a more hydrophilic ^ subunit via the N-glycosylated y subunit. During the passage through the secretion pathway, the signal sequence is removed by a signal peptidase. The resulting protoxin is further processed by the Kex1 p / Kex2p endoproteases found in the Golgi apparatus, so that finally an active toxin consisting of an α and β subunit, which are connected by a disulfide bridge, is secreted.
Fig. 2: Schematische Darstellung der Expressionsvektoren pREP-K28 und pINT-2: Schematic representation of the expression vectors pREP-K28 and pINT-
K28.K28.
Der Bereich des Spalthefen nm-^-Promotors für die Transkription-Initiation ist mitThe area of the gap yeast nm - ^ - promoter for the transcription initiation is with
Pnmtl und der für die Transkription-Termination mit Tnmtl gekennzeichnet. S/P symbolisiert die Prozessierungs- und Sekretionssequenz des K28 Killertoxins. DickePnmtl and the one marked for transcription termination with Tnmtl. S / P symbolizes the processing and secretion sequence of the K28 killer toxin. thickness
Linien repräsentieren Sequenzen aus Hefe, dünne Linien stellen Escherichia coli pUC19-Sequenzen dar (oriE, E. coli Replikationsursprung; AmpR, ß-Lactamase Gen; arsl, autonome replizierende Sequenz aus S. pombe; LEU2, S. cerevisiae LEU2-Gen; S. pombe ura4+-und Ieυ1*-Gene).Lines represent sequences from yeast, thin lines represent Escherichia coli pUC19 sequences (oriE, E. coli origin of replication; AmpR, β-lactamase Gene; arsl, autonomous replicating sequence from S. pombe; LEU2, S. cerevisiae LEU2 gene; S. pombe ura4 + and Ieυ1 * genes).
Fig. 3: Thiamin-regulierte Toxin-Expression der rekombinanten S. pombe Stämme. Gezeigt ist der Filter eines Western Blots, auf dem das aktive Toxin mit einem polyklonalem Antiserum gegen die ß-Untereinheit detektiert wird. Spuren 1 und 3, Kulturüberstände reprimierter S. pombe (pREP-K28 bzw. plNT-K28) Kulturen; Spuren 2 und 4, Kulturüberstände induzierter S. pombe (pREP-K28 bzw. plNT-K28) Kulturen; Spuren R und I (negative Kontrolle), Überstände zweier S. pombe Transformanden, die entweder nur das pREP1- oder das plNT5-Plasmid tragen; Spur C Positivkontrolle, teilweise aufgereinigtes reifes K28 Toxin; Spur S, vorgefärbte Proteinfraktion zur Molekulargewichtsbestimmung. Der große Pfeil markiert das 21 kDa schwere aktive, heterodimere Toxin; die beiden kleinen Pfeile markieren das sich in einem SDS-PAGE unter nicht-reduzierenden Bedingungen spontan bildende tetramere Derivat (α/ß)2 bzw. die monomere ß-Untereinheit.Fig. 3: Thiamine-regulated toxin expression of the recombinant S. pombe strains. The filter of a Western blot is shown, on which the active toxin is detected with a polyclonal antiserum against the β-subunit. Lanes 1 and 3, culture supernatants of repressed S. pombe (pREP-K28 and plNT-K28) cultures; Lanes 2 and 4, culture supernatants of induced S. pombe (pREP-K28 and plNT-K28) cultures; Lanes R and I (negative control), supernatants of two S. pombe transformants which carry either only the pREP1 or the plNT5 plasmid; Lane C positive control, partially purified mature K28 toxin; Lane S, pre-stained protein fraction for molecular weight determination. The large arrow marks the 21 kDa active, heterodimeric toxin; the two small arrows mark the tetramer derivative (α / ß) 2 which forms spontaneously in an SDS-PAGE under non-reducing conditions or the monomeric ß subunit.
Fig. 4: Vergleich der rekombinanten bzw. homologen Toxin-Sekretion in S. pombe bzw. S. cerevisiae.Fig. 4: Comparison of the recombinant or homologous toxin secretion in S. pombe or S. cerevisiae.
Gezeigt ist der Filter eines Western Blots, auf dem das aktive Toxin mit einem polyklonalen Antiserum gegen die ß-Untereinheit detektiert wird (Fig. 4a). Das Auftragsschema ist wie folgt: Spuren 1 und 2, extrazelluläre Proben je einer induzierten Kultur des S. pombe Stammes, der das Toxin von dem episomal vorliegendem Plasmid (pREP-K28) bzw. dem chromosomal integriertem Plasmid sekretiert (plNT-K28); Spur 3 extrazelluläre Proben des S. cerevisiae Stammes, der das K28-ppfox-Genprodukt episomal exprimiert (pFR5-TPI) bzw. des Virusinfizierten S. cerevisae Stammes MS 300c (s/ 2-2), der das Killertoxin überexprimiert. Spur 4, Positivkontrolle, konzentrierte und teilweise gereinigte Toxinfraktion einer S. cerevisiae K28 Killerhefe. Der große Pfeil markiert das 21 kDa schwere aktive, heterodimere Toxin; die beiden kleinen Pfeile markieren das sich in einer SDS-PAGE unter nicht-reduzierenden Bedingungen spontan bildende tetramere Derivat (α/ß)2 bzw. die monomere ß-Untereinheit. Fig. 4b zeigt die relative Toxin-Sekretion der Hefestämme anhand des Vergleiches der Toxin-Signale mittels eines Laser-Scan-Densitometers. Fig. 5: Schematische Darstellung der Expressionsvektoren pTZα/ßGFP und pTZsp- GFP.The filter of a Western blot is shown, on which the active toxin is detected with a polyclonal antiserum against the β-subunit (FIG. 4a). The application scheme is as follows: lanes 1 and 2, extracellular samples each of an induced culture of the S. pombe strain, which secretes the toxin from the episomally present plasmid (pREP-K28) or the chromosomally integrated plasmid (plNT-K28); Lane 3 extracellular samples of the S. cerevisiae strain, which expresses the K28-ppfox gene product episomally (pFR5-TPI) or the virus-infected S. cerevisae strain MS 300c (s / 2-2), which overexpresses the killer toxin. Lane 4, positive control, concentrated and partially purified toxin fraction from a S. cerevisiae K28 killer yeast. The large arrow marks the 21 kDa active, heterodimeric toxin; the two small arrows mark the tetramer derivative (α / β) 2 which forms spontaneously in an SDS-PAGE under non-reducing conditions or the monomeric β-subunit. 4b shows the relative toxin secretion of the yeast strains on the basis of the comparison of the toxin signals by means of a laser scan densitometer. Fig. 5: Schematic representation of the expression vectors pTZα / ßGFP and pTZsp-GFP.
Der Bereich des Spalthefen πm^-Promotors für die Transkription-Initiation ist mit Pnmtl und der für die Transkription-Termination mit Tnmtl gekennzeichnet. S/P symbolisiert die Prozessierungs- und Sekretionssequenz des K28 Killertoxins, GFP das grün-fluoreszierende Protein aus Aequorea victoria. Dicke Linien repräsentieren Sequenzen aus Hefe, dünne Linien stellen Escherichia coli pUC19-Sequenzen dar (oriE, E. coli Replikationsursprung; AmpR, ß-Lactamase Gen; arsl, autonome replizierende Sequenz aus S. pombe; .URA4, S. cerevisiae).The area of the gap yeast πm ^ promoter for the transcription initiation is marked with Pnmtl and that for the transcription termination with Tnmtl. S / P symbolizes the processing and secretion sequence of the K28 killer toxin, GFP the green fluorescent protein from Aequorea victoria. Bold lines represent sequences from yeast, thin lines represent Escherichia coli pUC19 sequences (oriE, E. coli origin of replication; AmpR, β-lactamase gene; arsl, autonomous replicating sequence from S. pombe; .URA4, S. cerevisiae).
Fig. 6/7: Sekretion heterologer Fusionsproteine in S. pombe. Gezeigt sind Filter von Western Blots, auf denen die Sekretion der jeweiligen Fusionen mit einem gegen das GFP-Protein gerichteten spezifischen Antikörper nachgewiesen wird. Die Belegung der beiden Filter ist die gleiche, wobei Figur 6 die GFP-Sekretion vom Plasmid pTZα/ß, und Figur 7 die GFP-Sekretion vom Plasmid pTZsp zeigt.Fig. 6/7: Secretion of heterologous fusion proteins in S. pombe. Filters from Western blots are shown, on which the secretion of the respective fusions with a specific antibody directed against the GFP protein is detected. The assignment of the two filters is the same, with FIG. 6 showing the GFP secretion from plasmid pTZα / β and FIG. 7 showing GFP secretion from plasmid pTZsp.
Spuren 1 und 3; Kulturüberstände reprimierter S. pomi e-Kulturen, die entweder als negative Kontrolle nur das Plasmid allein bzw. das Expressionsplasmid zur Sekretion des GFP tragen. Spuren 2 und 4; Kulturüberstände induzierter S. pombe Kulturen, die entweder als negative Kontrolle nur das Plasmid allein bzw. das Expressionsplasmid zur Sekretion des GFP tragen; Spur S, vorgefärbte Proteinfraktion zur Molekulargewichtsbestimmung; Spur C, Positivkontrolle, aufgereinigtes rekombinantes GFP Protein.Lanes 1 and 3; Culture supernatants of repressed S. pomi e cultures which either carry only the plasmid alone or the expression plasmid for secretion of the GFP as a negative control. Lanes 2 and 4; Culture supernatants of induced S. pombe cultures which either carry only the plasmid alone or the expression plasmid for secretion of the GFP as a negative control; Lane S, pre-stained protein fraction for molecular weight determination; Lane C, positive control, purified recombinant GFP protein.
Im folgenden sind einige Ausführungsbeispiele beschrieben ohne die Erfindung darauf zu beschränken.Some exemplary embodiments are described below without restricting the invention thereto.
Expression rekombinanter Killertoxine in S. pombeExpression of recombinant killer toxins in S. pombe
Die Plasmide zur episomalen bzw. chromosomal-integrierten Expression des K28 Killertoxins sind in Fig. 2 schematisch dargestellt. Zur Generierung wurde ein 1048 bp langes Xhol/Bglll-Fragment des K28 Killertoxin-kodierenden Hefe-Plasmids PPGK-M28-1 (Schmitt, M.J. und Tipper, DJ. (1995) Virology 213: 341-351 ) in die mit Sall/BamHI restringierten und linearisierten Expressionvektoren pREP1 und pINT5 aus S. pombe einkloniert. In beiden Konstrukten können damit die zu exprimierenden Proteine über den Thiamin-regulierbaren Promotor nmtl kontrolliert werden. Die Vektoren wurden darauf jeweils in die beiden Stämme der Wirtshefen S. pombe mittels Lithiumacetat-Methode transformiert. Die Transformanden wurden auf Minimalmedium ohne Uracil (bei plNT-K28) bzw. ohne Leucin (bei pREP-K28) selektiert. PINT-K28 bzw. pREP-K28 positive Hefetransformanden wurden auf ihren Killer-Phänotyp in einem Standardtest, dem Agar-Diffusions-Assay, auf Methylenblau gefärbten Agarplatten mit dem K28 toxinsensitiven S. cerevisae Stamm 192.2d getestet (Daten nicht gezeigt).The plasmids for the episomal or chromosomal-integrated expression of the K28 killer toxin are shown schematically in FIG. 2. A 1048 bp Xhol / BglII fragment of the K28 killer toxin-encoding yeast plasmid was used for generation PPGK-M28-1 (Schmitt, MJ and Tipper, DJ. (1995) Virology 213: 341-351) was cloned into the expression vectors pREP1 and pINT5 from S. pombe restricted and linearized with Sall / BamHI. In both constructs the proteins to be expressed can be controlled via the thiamine-regulable promoter nmtl. The vectors were then transformed into the two strains of the host yeast S. pombe using the lithium acetate method. The transformants were selected on minimal medium without uracil (for plNT-K28) or without leucine (for pREP-K28). PINT-K28 and pREP-K28 positive yeast transformants were tested for their killer phenotype in a standard test, the agar diffusion assay, on methylene blue stained agar plates with the K28 toxin-sensitive S. cerevisae strain 192.2d (data not shown).
Über einen Western Blot konnte die induzierbare Toxin-Sekretion der rekombinanten S. pombe Stämme nachgewiesen werden (Fig. 3).The inducible toxin secretion of the recombinant S. pombe strains could be demonstrated by a Western blot (FIG. 3).
Hierfür wurden jeweils 600 μl Zellkulturüberstand des S. pombe Stammes mit dem episomal vorliegenden Plasmid (pREP-K28) bzw. des S. pombe Stammes mit dem chromosomal vorliegenden Plasmid (plNT-K28) bei einer Dichte von 5 x 107 Zellen pro ml mit reprimierendem (Thiamin-haltigem, 25 μM) oder induzierendem (Thiamin- freiem) Medium abgenommen, Ethanol gefällt und unter nicht-reduzierenden Bedingungen auf einem 10-22.5 %igen Gradientengel (SDS-PAGE) aufgetrennt. Nach dem Blot auf eine PVDF-Membran wurde das aktive Toxin durch ein polyklonales Kaninchenantiserum (Primärantikörper) gegen die ß-Untereinheit des aktiven Toxins detektiert (Sekundärantikörper: Ziege anti Kaninchen IgG-Alkalische Phosphatase). Nach Inkubation der Membran mit einer NBT/BCIP-Färbelösung konnte die ß-Untereinheit des Toxins über eine Färbereaktion sichtbar gemacht werden.For this purpose, 600 μl of cell culture supernatant from the S. pombe strain with the episomal plasmid (pREP-K28) or the S. pombe strain with the chromosomal plasmid (plNT-K28) at a density of 5 × 10 7 cells per ml were used repressing (thiamine-containing, 25 μM) or inducing (thiamine-free) medium removed, ethanol precipitated and separated under non-reducing conditions on a 10-22.5% gradient gel (SDS-PAGE). After blotting on a PVDF membrane, the active toxin was detected by a polyclonal rabbit antiserum (primary antibody) against the β-subunit of the active toxin (secondary antibody: goat anti rabbit IgG alkaline phosphatase). After incubation of the membrane with an NBT / BCIP staining solution, the ß-subunit of the toxin could be made visible through a staining reaction.
Bestimmung der Toxin-Sekretion eines K28 Virus-infizierten S. cerevisiae Stammes und der rekombinanten S. pombe Stämme.Determination of the toxin secretion of a K28 virus-infected S. cerevisiae strain and the recombinant S. pombe strains.
Zur semiquantitativen Bestimmung der extrazellulären Toxinmengen wurden, wie in Beispiel 1 beschrieben, je 600 μl zellfreie Kulturüberstände von einer induzierenden (Thiamin-freien) Kultur des S. pombe Stammes, der das ppfox-Gen episomal (pREP- K28) enthält und eines S. pombe Stammes, der das ppfox-Gen chromosomal (pINT- K28) enthält, genommen und mit je 600 μl zellfreien Kulturüberständen eines K28 infizierten überexprimierenden S. cerevisiae MS 300c (s/f/2-2)-Stammes und einem K28-Toxin episomal exprimierenden S. cerevisiae pFR5-TPI-Stammes verglichen (Fig. 4a). Diese Proben wurden Ethanol gefällt und unter nicht-reduzierenden Bedingungen durch SDS-PAGE aufgetrennt. Die spezifische Detektion des aktiven Toxins wurde wiederum mit einem polyklonalen Antikörper gegen die ß-Untereinheit durchgeführt (siehe Bsp. 1). Über die densitometrische Auswertung der gefärbten Banden mit einem Laser Scanner konnten die relativen Expressionstärken miteinander verglichen werden (Fig. 4b).For the semi-quantitative determination of the extracellular amounts of toxin, as described in Example 1, 600 μl of cell-free culture supernatants from an inducing (thiamine-free) culture of the S. pombe strain, which contains the episomal ppfox gene (pREP-K28) and one S. pombe strain that contains the ppfox gene chromosomal (pINT- K28), taken and compared with 600 μl cell-free culture supernatants of a K28 infected overexpressing S. cerevisiae MS 300c (s / f / 2-2) strain and a K28 toxin episomal expressing S. cerevisiae pFR5-TPI strain (Fig 4a). These samples were ethanol precipitated and separated by SDS-PAGE under non-reducing conditions. The specific detection of the active toxin was again carried out using a polyclonal antibody against the β-subunit (see Example 1). The relative expression levels could be compared with one another by means of the densitometric evaluation of the colored bands with a laser scanner (FIG. 4b).
Killertoxin-vermittelte Sekretion heterologer Proteine in S. pombe.Killer toxin-mediated secretion of heterologous proteins in S. pombe.
Die Konstrukte zur episomalen Sekretion des grünen-fluoreszierenden-Proteins (GFP) sind in Fig. 5 schematisch dargestellt. Eine DNA, die für das GFP Protein aus Aequorea victoria kodiert wurde als BamHI/BamHI-Fragment mit einem Bglll/Bglll- Fragment des Expressionsvektors pTZα/ß bzw. mit einem Bglll/Bglll-Fragment des Expressionsvektors pTZsp kloniert. Die Hybridplasmide pTZα/ß und pTZsp sind aus Sequenzen des Expressionsvektors pREP4x und dem K28 ppfox-Gen bzw. des Expressiönsvektors pREP4x und der Signalsequenz S/P des ppfox-Gens entstanden (Basi, G., Schmid, E., Maundrell, K. (1993) Gene 123: 131-136; Schmitt, M.J. und Tipper, DJ. (1995) Virology 213: 341-351).The constructs for the episomal secretion of the green fluorescent protein (GFP) are shown schematically in FIG. 5. A DNA which was encoded for the GFP protein from Aequorea victoria was cloned as a BamHI / BamHI fragment with a Bglll / Bglll fragment of the expression vector pTZα / β or with a Bglll / Bglll fragment of the expression vector pTZsp. The hybrid plasmids pTZα / ß and pTZsp resulted from sequences of the expression vector pREP4x and the K28 ppfox gene or the expression vector pREP4x and the signal sequence S / P of the ppfox gene (Basi, G., Schmid, E., Maundrell, K. (1993) Gene 123: 131-136; Schmitt, MJ and Tipper, DJ. (1995) Virology 213: 341-351).
Im ersten Fall wird damit ein Fusionsprotein, bestehend aus der α-, und der ß- Untereinheit des aktiven Toxins und dem GFP-Protein (pTZ /ß-GFP), in dem anderen Fall eine Fusion bestehend aus der Signalsequenz (S/P) und dem GFP- Protein exprimiert (pTZsp-GFP). Nach Prozessierung und Sekretion sollte man u.a. das rekombinante GFP-Protein im Überstand nachweisen können. Die Plasmide wurden mittels Lithiumacetat-Methode in S. pombe transformiert und auf Minimalmedium ohne Uracil selektioniert. In beiden Stämmen werden die zu exprimierenden Proteine wiederum über den Thiamin-regulierten Promotor nmtl kontrolliert.In the first case, a fusion protein consisting of the α and the β subunit of the active toxin and the GFP protein (pTZ / β-GFP), in the other case a fusion consisting of the signal sequence (S / P) and the GFP protein expressed (pTZsp-GFP). After processing and secretion, one should can detect the recombinant GFP protein in the supernatant. The plasmids were transformed into S. pombe using the lithium acetate method and selected on minimal medium without uracil. In both strains, the proteins to be expressed are in turn controlled via the thiamine-regulated promoter nmtl.
Für die aufgeführten Experimente wurden die rekombinanten Stämme zuerst 24 Stunden in Uracil-haltigem EEM-Medium und danach zur Repression 24 Stunden im gleichen Medium in Gegenwart von 25 μM Thiamin kultiviert (Fig. 6/7). Zur nachfolgenden Derepression und somit Expression der Fusionsproteine wurden dieFor the experiments listed, the recombinant strains were first cultured for 24 hours in uracil-containing EEM medium and then for repression in the same medium for 24 hours in the presence of 25 μM thiamine (Fig. 6/7). to subsequent depression and thus expression of the fusion proteins were the
Kulturen für 36 Stunden in Thiamin-freiem EEM-Medium inkubiert.Cultures incubated for 36 hours in thiamine-free EEM medium.
Von den einzelnen Zellkulturen wurden 4.5 ml Kulturüberstand lyophilisiert, in 30μl4.5 ml of the culture supernatant from the individual cell cultures were lyophilized, in 30 μl
H2O resuspendiert und gelelektrophoretisch auf einem reduzierenden SDS-PAGE analysiert. Nach Blot auf eine PVDF-Membran konnte sekretiertes GFP mittels einerH 2 O resuspended and analyzed by gel electrophoresis on a reducing SDS-PAGE. After blotting on a PVDF membrane, secreted GFP could be determined using a
Antikörperreaktion nachgewiesen werden (spezifischen Primärantikörper: anti GFPAntibody response can be detected (specific primary antibody: anti GFP
IgG der Maus; Sekundärantikörper. anti-Maus IgG-Peroxidase).Mouse IgG; Secondary antibody. anti-mouse IgG peroxidase).
Durch Inkubation der Membran mit dem POD-BM Chemilumineszenz Blotting-By incubating the membrane with the POD-BM chemiluminescence blotting
Substrat der Firma Röche Diagnostics konnte die entstehende Chemilumineszenz mittels Exposition auf einem Röntgenfilm sichtbar gemacht werden. The resulting chemiluminescence could be visualized by exposure to an X-ray film using the Röche Diagnostics substrate.
Literaturhinweise zum Stand der TechnikReferences to the state of the art
Basi, G., Schmid, E., Maundrell, K. (1993) Gene 123: 131-136.Basi, G., Schmid, E., Maundrell, K. (1993) Gene 123: 131-136.
Broker, M., Ragg , H., Karges, H.E. (1987) Biochemical Biophysical Acta 908: 203- 213.Broker, M., Ragg, H., Karges, H.E. (1987) Biochemical Biophysical Acta 908: 203-213.
Lu, Y. Bauer, J.C., Greener, A. (1997) Gene 200: 135-144.Lu, Y. Bauer, J.C., Greener, A. (1997) Gene 200: 135-144.
Sambamurti, K. (1997) In: Foreign gene expression in fission Yeast: S. pombe, Giga- Hama, Y. und Kumagai, H. (eds.) Springer Verlag: 149-158.Sambamurti, K. (1997) In: Foreign gene expression in fission Yeast: S. pombe, Giga-Hama, Y. and Kumagai, H. (eds.) Springer Verlag: 149-158.
Schmitt, M.J. und Tipper, D.J. (1990) Molecular and Cellular Biology 10: 4807-4815.Schmitt, M.J. and Tipper, D.J. (1990) Molecular and Cellular Biology 10: 4807-4815.
Schmitt, M.J. und Tipper, D.J. (1995) Virology 213: 341-351.Schmitt, M.J. and Tipper, D.J. (1995) Virology 213: 341-351.
Tokunaga, M., Kawamura, A., Yonekyu, S. (1993) Yeast 9: 379-387. Tokunaga, M., Kawamura, A., Yonekyu, S. (1993) Yeast 9: 379-387.

Claims

Patentansprüche: claims:
1. Sekretionssignalsequenz für eukaryotische Expressionssyteme mit der Aminosäuresequenz Seq. Id. No.1 oder einer funktionellen Variante davon.1. Secretion signal sequence for eukaryotic expression systems with the amino acid sequence Seq. Id. No.1 or a functional variant of it.
2. Sekretionssignalsequenz für eukaryotische Expressionssyteme mit einer Aminosäuresequenz die mindestens 80% homolog zu der Aminosäuresequenz Seq. Id. No.1 ist.2. Secretion signal sequence for eukaryotic expression systems with an amino acid sequence which is at least 80% homologous to the amino acid sequence Seq. Id. No.1 is.
3. Sekretionssignalsequenz für eukaryotische Expressionssyteme die eine mindestens 20 Aminosäuren lange Teilsequenz gemäß der Aminosäuresequenz Seq. Id. No.1 enthält.3. Secretion signal sequence for eukaryotic expression systems which have a partial sequence of at least 20 amino acids according to the amino acid sequence Seq. Id. No.1 contains.
4. DNA-Sequenz, die für ein Sekretionssignal gemäß der Ansprüche 1 bis 3 kodiert.4. DNA sequence which codes for a secretion signal according to claims 1 to 3.
5. DNA-Sequenz gemäß Anspruch 4 mit einer Nukleotidsequenz Seq. Id. No. 2 oder einer funktionellen Variante davon.5. DNA sequence according to claim 4 with a nucleotide sequence Seq. Id. No. 2 or a functional variant thereof.
6. Expressionsvektoren für eukaryotische Zellen enthaltend einen Promotor und die S/P-Sekretionssignalsequenz des ppfox-Gens des Virus K28 gemäß Seq. Id. No. 2 oder einer funktionellen Variante dieser Sequenz mit einer Sequenzhomologie von mindestens 70%, die 3 -wärts zum Promotor liegt.6. Expression vectors for eukaryotic cells containing a promoter and the S / P secretion signal sequence of the ppfox gene of the virus K28 according to Seq. Id. No. 2 or a functional variant of this sequence with a sequence homology of at least 70% which lies 3 -wards to the promoter.
7. Expressionsvektoren für eukaryotische Zellen gemäß Anspruch 6 enthaltend eine funktionelle Variante der S/P-Sekretionssequenz, die unter stringenten Bedingungen mit der S/P-Sekretionssignalsequenz (Seq. Id. No. 2) hybridisiert oder eine dazu allele Variante.7. Expression vectors for eukaryotic cells according to claim 6 containing a functional variant of the S / P secretion sequence which hybridizes under stringent conditions with the S / P secretion signal sequence (Seq. Id. No. 2) or an allele variant thereof.
8. Expressionsvektoren für eukaryotische Zellen gemäß einem der Ansprüche 6 oder 7, dadurch gekennzeichnet, dass der Expressionsvektor zusätzlich die a- und/oder ß- und/oder ^-Untereinheit des K28-ppfox-Gens oder Teile davon enthält.8. Expression vectors for eukaryotic cells according to one of claims 6 or 7, characterized in that the expression vector additionally the a- and / or β and / or ^ subunit of the K28 ppfox gene or parts thereof.
9. Expressionsvektor für eukaryotische Zellen gemäß Anspruch 8, dadurch gekennzeichnet, dass der Expressionsvektor die zum Spleißen des K28-ppfox- Gentranskripts nötigen Sequenzbereiche des ppfox-Gens oder eine funktionelle Variante davon mit einer Homologie von mindestens 70% enthält.9. Expression vector for eukaryotic cells according to claim 8, characterized in that the expression vector contains the sequence regions of the ppfox gene necessary for splicing the K28-ppfox gene transcript or a functional variant thereof with a homology of at least 70%.
10. Expressionsvektor für eukaryotische Zellen gemäß einem der Ansprüche 6 bis 9, dadurch gekennzeichnet, dass der Expressionsvektor ein 3'-wärts zum Promotor liegende kodierende Nukleinsäuresequenz enthält.10. Expression vector for eukaryotic cells according to one of claims 6 to 9, characterized in that the expression vector contains a coding nucleic acid sequence lying 3 ' towards the promoter.
11. Expressionsvektor für eukaryotische Zellen gemäß Anspruch 10, dadurch gekennzeichnet, dass das die kodierende Nukleinsäuresequenz ein heterologes Gen ist.11. Expression vector for eukaryotic cells according to claim 10, characterized in that the coding nucleic acid sequence is a heterologous gene.
12. Expressionsvektor für eukaryotische Zellen gemäß Anspruch 10 oder 11 , dadurch gekennzeichnet, dass das die kodierende Nukleinsäuresequenz ein eukaryotisches Gen ist.12. Expression vector for eukaryotic cells according to claim 10 or 11, characterized in that the coding nucleic acid sequence is a eukaryotic gene.
13. Expressionsvektor für eukaryotische Zellen gemäß einem der Ansprüche 6 bis 12, dadurch gekennzeichnet, dass die Expressionsvektoren weitere Nukleinsäuresequenzen enthalten, die für Selektionsmarker, Repressoren oder Aktivatoren kodieren und/oder Nukleinsäuresequenzen enthalten, die als Replikationsstartpunkt, Terminationssignal oder Operator wirken.13. Expression vector for eukaryotic cells according to one of claims 6 to 12, characterized in that the expression vectors contain further nucleic acid sequences which code for selection markers, repressors or activators and / or contain nucleic acid sequences which act as a starting point of replication, termination signal or operator.
14. Expressionsvektor für eukaryotische Zellen gemäß einem der Ansprüche 6 bis 13 dadurch gekennzeichnet, dass der Expressionsvektor den durch Thiamin induzierbaren Promotor nmt 1 enthält.14. Expression vector for eukaryotic cells according to one of claims 6 to 13, characterized in that the expression vector contains the thiamine-inducible promoter nmt 1.
15. Expressionsvektor für eukaryotische Zellen gemäß Anspruch 13 dadurch gekennzeichnet, dass der Expressionsvektor als Terminationssignal die Tnmtl - Nukleinsäuresequenz enthält. 15. Expression vector for eukaryotic cells according to claim 13, characterized in that the expression vector contains the Tnmtl nucleic acid sequence as a termination signal.
16. Expressionsvektor für eukaryotische Zellen gemäß einem der Ansprüche 6 bis16. Expression vector for eukaryotic cells according to one of claims 6 to
15, dadurch gekennzeichnet, dass der Expressionsvektor ein pREP-K28-Vektor, ein plNT-K28-Vektor, ein pTZ / -Vektor oder ein pTZsp-Vektor ist.15, characterized in that the expression vector is a pREP-K28 vector, a plNT-K28 vector, a pTZ / vector or a pTZsp vector.
17. Expressionssystem umfassend eine eukaryotische Zelle und einen Expressionsvektor gemäß einem der Ansprüche 10 bis 16.17. Expression system comprising a eukaryotic cell and an expression vector according to one of claims 10 to 16.
18. Expressionssystem gemäß Anspruch 17, dadurch gekennzeichnet, dass die eukaryotische Zelle eine Hefe ist.18. Expression system according to claim 17, characterized in that the eukaryotic cell is a yeast.
19. Expressionssystem gemäß Anspruch 18, dadurch gekennzeichnet, dass die Hefezelle aus der Gruppe Schizosaccharomyces pombe, Saccharomyces cerevisiae ausgewählt ist.19. Expression system according to claim 18, characterized in that the yeast cell is selected from the group Schizosaccharomyces pombe, Saccharomyces cerevisiae.
20. Fusionsproteine enthaltend ein S/P-Sekretionssignal (Seq. ID. No. 1) oder eine funktionelle Variante davon mit einer Sequenzhomologie von mindestens 80%.20. Fusion proteins containing an S / P secretion signal (Seq. ID. No. 1) or a functional variant thereof with a sequence homology of at least 80%.
21. Fusionsproteine enthaltend das S/P-Sekretionssignal (Seq. ID. No. 1) oder eine funktionellen Variante davon mit einer Sequenzhomologie von mindestens 80% erhältlich durch Expression eines Gens, das 3'-wärts zum Promotor eines Expressionsvektors gemäß Anspruch 4 kloniert ist.21. Fusion proteins containing the S / P secretion signal (Seq. ID. No. 1) or a functional variant thereof with a sequence homology of at least 80% obtainable by expression of a gene which clones 3 ' towards the promoter of an expression vector according to claim 4 is.
22. DNA-Sequenzen kodierend für ein Fusionsprotein gemäß den Ansprüchen 20 und 21.22. DNA sequences coding for a fusion protein according to claims 20 and 21.
23. Verwendung eines Expressionsvektors gemäß einem der Ansprüche 6 bis 16 zur Klonierung von Genen.23. Use of an expression vector according to one of claims 6 to 16 for cloning genes.
24. Verwendung eines Expressionsvektors gemäß Anspruch 10 zur Transformation von eukaryotischen Zellen. 24. Use of an expression vector according to claim 10 for the transformation of eukaryotic cells.
5. Verwendung eines Expressionssystems gemäß Anspruch 17 zur Herstellung von Proteinen. 5. Use of an expression system according to claim 17 for the production of proteins.
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