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WO2002048187A2 - Peptides du signal de secretion, leurs sequences adn, vecteurs d'expression pour cellules eucaryotes preparees a partir de ceux-ci, et leur utilisation pour la preparation biotechnologique de proteines - Google Patents

Peptides du signal de secretion, leurs sequences adn, vecteurs d'expression pour cellules eucaryotes preparees a partir de ceux-ci, et leur utilisation pour la preparation biotechnologique de proteines Download PDF

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Publication number
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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PCT/EP2001/014588
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German (de)
English (en)
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WO2002048187A3 (fr
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Manfred Schmitt
Uwe WÖLK
Peter Wagner
Tatjana Zagorc
Tanja Heintel
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Phylos, Inc.
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Priority to AU2002235771A priority Critical patent/AU2002235771A1/en
Publication of WO2002048187A2 publication Critical patent/WO2002048187A2/fr
Publication of WO2002048187A3 publication Critical patent/WO2002048187A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—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.

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Abstract

L'invention concerne des séquences peptidiques du signal de sécrétion, ainsi que des acides nucléiques codant pour celles-ci, des vecteurs d'expression pour des cellules eucaryotes, les constructions cellulaires transfixées avec ces vecteurs, ainsi que l'utilisation de vecteurs et de constructions cellulaires pour la fabrication efficace, par biotechnologie, de protéines avec des cellules eucaryotes.
PCT/EP2001/014588 2000-12-14 2001-12-12 Peptides du signal de secretion, leurs sequences adn, vecteurs d'expression pour cellules eucaryotes preparees a partir de ceux-ci, et leur utilisation pour la preparation biotechnologique de proteines WO2002048187A2 (fr)

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AU2002235771A AU2002235771A1 (en) 2000-12-14 2001-12-12 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

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DE10062302.6 2000-12-14
DE2000162302 DE10062302A1 (de) 2000-12-14 2000-12-14 Sekretionssignalpeptide, deren DNA-Sequenzen, damit herstellbare Expressionsvektoren für eukaryotische Zellen und deren Verwendung zur biotechnologischen Herstellung von Proteinen

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Cited By (5)

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EP2617732A1 (fr) 2012-01-19 2013-07-24 Vib Vzw Outils et procédés pour l'expression de protéines de membrane
US8815580B2 (en) 2008-08-08 2014-08-26 Vib Vzw Cells producing glycoproteins having altered glycosylation patterns and method and use thereof
WO2015032899A1 (fr) 2013-09-05 2015-03-12 Vib Vzw Cellules produisant des molécules contenant fc et présentant des motifs de glycosylation modifiés et leurs procédés d'utilisation
GB201901608D0 (en) 2019-02-06 2019-03-27 Vib Vzw Vaccine adjuvant conjugates
US11293012B2 (en) 2015-07-09 2022-04-05 Vib Vzw Cells producing glycoproteins having altered N- and O-glycosylation patterns and methods and use thereof

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DE10252245A1 (de) * 2002-11-07 2004-05-27 Prof. Dr. Danilo Porro Università degli Studi di Milano-Bicocca Dipartimento die Biotechnologie e Bioscienze Verfahren zur Expression und Sekretion von Proteinen mittels der nicht-konventionellen Hefe Zygosaccharomyces bailii

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DE69732378D1 (de) * 1997-05-27 2005-03-03 Hanil Synthetic Fiber Co Ltd Verfahren zur herstellung von rekombinanten eiweissen durch verwendung von hocheffizienten expressionsvektoren aus saccharomyces cerevisiae

Cited By (10)

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US8815580B2 (en) 2008-08-08 2014-08-26 Vib Vzw Cells producing glycoproteins having altered glycosylation patterns and method and use thereof
EP2617732A1 (fr) 2012-01-19 2013-07-24 Vib Vzw Outils et procédés pour l'expression de protéines de membrane
WO2013107905A1 (fr) 2012-01-19 2013-07-25 Vib Vzw Outils et méthodes utilisés pour l'expression de protéines membranaires
US9890217B2 (en) 2012-01-19 2018-02-13 Vib Vzw Tools and methods for expression of membrane proteins
US11479791B2 (en) 2012-01-19 2022-10-25 Vib Vzw Tools and methods for expression of membrane proteins
WO2015032899A1 (fr) 2013-09-05 2015-03-12 Vib Vzw Cellules produisant des molécules contenant fc et présentant des motifs de glycosylation modifiés et leurs procédés d'utilisation
US10202590B2 (en) 2013-09-05 2019-02-12 Vib Vzw Cells producing Fc-containing molecules having altered glycosylation patterns and methods and use thereof
US11421209B2 (en) 2013-09-05 2022-08-23 Vib Vzw Cells producing Fc containing molecules having altered glycosylation patterns and methods and use thereof
US11293012B2 (en) 2015-07-09 2022-04-05 Vib Vzw Cells producing glycoproteins having altered N- and O-glycosylation patterns and methods and use thereof
GB201901608D0 (en) 2019-02-06 2019-03-27 Vib Vzw Vaccine adjuvant conjugates

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