EP1214425A1 - Regulatory sequences and expression cassettes for yeasts - Google Patents

Regulatory sequences and expression cassettes for yeasts

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Publication number
EP1214425A1
EP1214425A1 EP00965925A EP00965925A EP1214425A1 EP 1214425 A1 EP1214425 A1 EP 1214425A1 EP 00965925 A EP00965925 A EP 00965925A EP 00965925 A EP00965925 A EP 00965925A EP 1214425 A1 EP1214425 A1 EP 1214425A1
Authority
EP
European Patent Office
Prior art keywords
expression
seq
sequence
yeast
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00965925A
Other languages
German (de)
French (fr)
Inventor
Dietmar Becher
Rimantas Siekstele
Danguole Bartkeviciute
Kestutis Sasnauskas
Leopold DÖHNER
Salah Salim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TAD Pharma GmbH
Original Assignee
TAD PHARM WERK
TAD Pharmazeutisches Werk GmbH
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Filing date
Publication date
Application filed by TAD PHARM WERK, TAD Pharmazeutisches Werk GmbH filed Critical TAD PHARM WERK
Publication of EP1214425A1 publication Critical patent/EP1214425A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01015—Polygalacturonase (3.2.1.15)

Definitions

  • the invention relates to a DNA sequence which is active as a promoter in yeast cells, an expression and optionally secretion system containing the same, plasmids containing this system, host cells transformed with the DNA and methods for producing proteins and polypeptides.
  • K.marxianus can use a variety of carbon and energy sources for growth and is not very temperature sensitive.
  • Kluyveromyces maxrxianus can grow at temperatures up to 45 ° C and is therefore easier to grow than the temperature-sensitive Saccharo / 77yce $ strains.
  • the cells of fast growing K.marxian ⁇ s strains can divide every 35 minutes under optimal conditions.
  • the nucleotide sequence according to the invention comprises a sequence which is active as a promoter and provides an expression system which is very variable and is suitable for Kluyveromyces, but can also be used for other types of yeast, in particular Saccharomyces cerevisiae.
  • the DNA sequence according to the invention according to SEQ ID No. 1 is a nucleic acid sequence which contains regulatory regions of a gene which codes for the enzyme endopolygalacturonase.
  • the enzyme endopolygalacturonase breaks down pectin by breaking 1,4- ⁇ -D-galactosituronic bonds between two non-methylated galacturonic acid residues.
  • This enzyme occurs in yeast strains of the species Kluyveromyces marxian ⁇ s, among others.
  • the sequence of the endo- polygalacturonasegens from Kluyveromyces marxianus var. marxianus was published in Yeast 15, 31 1 -322 (1999). However, the promoter region that can be derived from the specified sequence did not reliably lead to the expression of various proteins.
  • SEQ ID No. 1 a sequence which comprises at least part of the nucleotides of SEQ ID No. 1, preferably at least nucleotides 1 to 1 134 and in particular the entire sequence, enables the expression of proteins in a very advantageous and reproducible manner .
  • the claimed sequence according to SEQ ID No. 1 has several regulatory components, so that it can perform its function as a promoter under the most varied of conditions.
  • the promoter according to the invention can be induced by adding pectin to the culture medium. This is advantageous since pectin is a readily available substance and therefore a cheap induction agent is available for the system according to the invention.
  • nucleic acid sequences with regulatory activity include those sequences which have arisen by modification, substitution, deletion or insertion or combinations thereof, which have the same or a better regulatory activity than the promoter, the terminator or the signal sequence.
  • sequence according to SEQ ID No. 1 can also be included with further regulatory upstream sequences with activator and / or reporter functions.
  • those sequences are furthermore considered which have a homology with the claimed nucleotides or sequences of at least 80%, more preferably at least 90% and in particular 95%, as long as they also have a comparable activity.
  • the sequence according to SEQ ID No. 1 is preferably provided in the form of an expression system or an expression cassette for the expression of proteins and peptides.
  • the expression cassette according to the invention comprises a regulatory sequence as shown in SEQ ID No. 1 or a part thereof active as a promoter, an insertion cloning site into which the polynucleotide for the protein to be expressed can be cloned, and the nucleotide sequence according to SEQ ID No.
  • the expression cassette can be used in many ways.
  • the insertion cloning site is an interface where the sequence can be cut open and the polynucleotide for the desired protein or peptide can be ligated in.
  • the protein is produced intracellularly after induction during expression and is not removed from the cell. After disruption of the cell, it can then be obtained in a manner known per se.
  • This embodiment is suitable both for small peptides and proteins that are unstable outside the cell and for proteins that are generally located intracellularly.
  • an expression and secretion system according to the invention is used.
  • this system comprises the nucleic acid sequence according to SEQ ID No. 1 or a part thereof active as a promoter, the sequence according to SEQ ID No. 2 as a terminator and, between these two sequences, the signal sequence according to SEQ ID No. 3
  • the cultivation is carried out in a manner known per se, wherein either the protein is continuously released into the medium in a continuous process and can be continuously obtained from the fermentation broth, or the cells are cultivated and harvested in a batch process and then the protein from the broth can be won.
  • the expression cassette according to the invention is suitable both for the expression of suitable autonomously replicating plasmids and for incorporation into yeast chromosomes via integrative vectors.
  • the invention further relates to the plasmids pEPG1-1, pEPG1 -2 and pEPGsec, which are explained in more detail in FIGS. 1 to 3 and which contain the expression systems according to the invention.
  • These plasmids are recombinant bacterial plasmids and can be used in the present form for the amplification of the expression cassettes.
  • the plasmids are contained in the microorganisms DSM 12919, DSM 12920, DSM 12921 or DSM 12922 * and are deposited with them.
  • the expression cassette with suitable restriction endonucleases for the interfaces at the edges of the expression cassette are intended to cut out and insert the expression cassette into a yeast vector.
  • the vectors usually contain selection markers in order to be able to select successfully transformed cells in a manner known per se.
  • the plasmids can optionally be propagated in f, coli and then used in Kluyveromyces marxianus or another Kl ⁇ yveromyces strain or also another yeast strain.
  • Known plasmids based on the Kluyveromyces droso- pKD1 can be used. Descendants of this plasmid are suitable for use in Kluyveromyces marxianus and, when using the expression system according to the invention, lead to effective expression and secretion of foreign proteins in the corresponding host.
  • the expression cassette including the polynucleotide to be expressed, can be cut out of the plasmids according to the invention and prepared as above and, as a linear or circularized DNA strand, can be brought into direct contact with yeast cells as an integration cassette in order to be taken up by them. Because of the homology with the endopolyglacturonase gene, the DNA is then taken up in part of the treated cells by exchanging it with the endogalacturonase gene. The selection of successfully transfected yeast
  • the expression cassette according to the invention is stably built into chromosomes and, if the cells are grown under optimal conditions, leads to a good yield of the desired protein.
  • the number of copies of the system can be adjusted depending on the type of protein or peptide to be expressed. Since the endopolygalacturonase gene is only present once in the yeast chromosome set, a transfection or transformation with the expression system provided according to the invention will also only have one copy of the expression vector per successfully transformed cell. If a higher number of copies is desired, sequences of a gene which is present in a larger number of copies in the chromosome set, e.g. for rDNA, ligated to cause a higher number of exchange events.
  • a marker is additionally incorporated into the sequence in a manner known per se, so that the successfully transformed cells can be selected. Methods and markers suitable for this are known to the person skilled in the art and do not require any further explanation here.
  • the system according to the invention is very variable.
  • only the sequence according to SEQ ID No. 1 or a part thereof active as a promoter can be combined with other nucleic acid sequences which provide further regulatory sequences and with a heterologous nucleotide sequence.
  • the sequence according to SEQ ID No. 1 or a part thereof active as a promoter can be combined with the sequence of SEQ ID No. 2 in order to provide a regulatory system which is homologous in Kluyveromyces marxianus and in which the polynucleotide for the protein to be expressed is used or a system of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.
  • Ki ⁇ yveromyces ZeWen can grow with many different C sources and are not very demanding in terms of other nutrients, and are also insensitive to temperature, a very effective system is provided here.
  • the reliable expression of the foreign proteins is achieved by the regulatory sequence of the invention provided according to the invention.
  • a system is provided which allows the promising yeast type Kluyveromyces marxianus to be used as a host due to its exceptional physiological performance.
  • the system according to the invention is suitable for the expression of peptides, polypeptides, proteins and hybrid molecules including glycosylated proteins.
  • the expression cassette can also contain the complete sequence of the endopolygalacturonase enzyme or parts thereof, a sequence for a desired protein being inserted between the endopolygalacturonase gene and the terminator sequence.
  • a hybrid is then obtained during expression, from which the endopolygalacturonase is split off in a manner known per se.
  • an "expression vector” is a DNA molecule that can be linear or circular and contains a segment that encodes a sequence for a protein or peptide of interest that is operatively linked to regulatory sequences. These regulatory sequences include at least promoter and terminator sequences.
  • the expression vector can additionally contain selectable markers and other regulatory elements and must enable the transfer and multiplication in host cells.
  • the expression vectors can be replicated autonomously or by integration into the host genome.
  • DNA or "polynucleotide” includes polymeric forms of deoxyribonucieotides and ribonucleotides of any length and any modification in single and double-stranded form.
  • operatively connected means that the individual segments are arranged so that they serve the intended purpose, i.e. can initiate transcription and can promote expression from the start of replication to the termination sequence.
  • the claimed sequences can have further short sequences that do not interfere with the biological activity of the molecule. Furthermore, the claimed sequences also include allelic variants of the sequence, ie alternative forms of the gene which have arisen from mutation.
  • allelic variants of the sequence ie alternative forms of the gene which have arisen from mutation.
  • the term "protein or peptide” refers to a molecular chain of amino acids with biological activity. The proteins and / or polypeptides can be modified in vivo or in vitro, for example by glycosylation and phosphorylation.
  • Hybrid molecules are understood to mean molecules which comprise both homologous parts and heterologous parts, for example those proteins which comprise a combination of endopolygalacturonase or parts thereof with a foreign protein, or for example nucleotide sequences in which DNA from Kluyveromyces marxianus is combined with DNA from other microorganisms ,
  • the expression cassette according to the invention is suitable, inter alia, for yeasts of the strains Kluyveromyces and Saccharomyces and is preferably used in yeast strains of the species Kluyveromyces marxianus var. Marxianus.
  • a particularly preferred strain with particularly favorable expression properties is Kluyveromyces marxianus var. Marxianus BKM Y-719, which was described by Siekstele et al. 1999 (Yeast 1 5, 31 1 to 322 (1999)).
  • Another object of the invention is a process for the production of a recombinant protein, which is characterized in that a yeast cell is transfected or transformed with an autonomously replicating plasmid which comprises an expression cassette according to the invention and a polynucleotide which encodes a foreign protein Yeast cell is grown under conditions suitable for the expression of the foreign protein and the protein wins.
  • the invention also relates to a method for producing a recombinant protein, which is characterized in that an expression cassette according to the invention is placed in a yeast cell, where the expression cassette is built into a chromosome, the cell is grown and then the protein is obtained.
  • the expression cassette according to the invention is particularly preferably used as a module which enables the construction of episomal or integrative expression vectors which contain the regulatory sequences according to SEQ ID No. 1, 2 and / or 3.
  • the expression system according to the invention is suitable for the expression of various heterologous proteins.
  • the system is particularly preferably used for the expression of HBVS antigen (hepatitis B virus, surface antigen) and virus protein 1 from polyoma virus. These proteins are antigenic proteins and can be used particularly advantageously as vaccines.
  • HBVS antigen hepatitis B virus, surface antigen
  • virus protein 1 from polyoma virus.
  • Figures 1 to 4 show plasmids with the expression cassettes according to the invention
  • Figure 6 shows the primers used in Example 2.
  • FIG. 1 shows the plasmid pEPG1-1.
  • This plasmid contains an expression cassette with the promoter according to the invention according to SEQ ID No. 1 (referred to as EPGI prom), an insertion cloning site which can be cut with BspT1, and the terminator sequence according to SEQ ID No. 2 (referred to as EPGIterm).
  • EPGI prom the promoter according to the invention according to SEQ ID No. 1
  • BspT1 an insertion cloning site which can be cut with BspT1
  • the terminator sequence according to SEQ ID No. 2 referred to as EPGIterm
  • Fig. 2 shows the plasmid pEPG1-2.
  • This plasmid contains the expression cassette according to the invention, which has a sequence according to SEQ ID No. 1 with nucleotides 572 to 1 134 (designated EPGI prom), which is active as a promoter, an insertion cloning site which can be cut with BspT1, and a terminator sequence, which comprises nucleotides 28 to 541 of SEQ ID No. 2 (referred to as EPGIterm).
  • the insertion cloning site replaces the open reading frame of the endopolygalacturonase gene including the sequences -1 to -12 and 1087 to 1115 of this gene.
  • Fig. 3 shows the plasmid pEPGsec.
  • This plasmid contains an expression and secretion system of the invention.
  • the plasmid contains an expression cassette with a promoter according to SEQ ID No. 1 (referred to as EPGI prom), a terminator according to SEQ ID No. 2 (referred to as EPGIterm), a signal sequence according to SEQ ID No. 3 (referred to as EOGI Iyd) and two interfaces to insert the desired nucleic acid sequence for the protein to be expressed.
  • This expression cassette was ligated into the multicloning site of the plasmid pUC19.
  • the open reading frame of the endopolygalacturonase gene was removed from positions 75 to 1084 and replaced by a linker with the cloning sites Eco 1471 and BpU 1 1021.
  • Figure 4 shows the plasmid pUC19PG.
  • a 2.198 kb PstI DNA restriction fragment from a recombinant LambdaGEM TM 12 bacteriophage from a Kluyveromyces marxianus genomic library was ligated into the PstI restriction site of the multicloning site of the plasmid pUC19.
  • 5 shows the plasmid pUC19-PG1 a.
  • a 2.735 kb Stul-Pvull DNA restriction fragment from a recombinant LambdaGEM TM 12 bacteriophage from a Kluyveromyces marxianus genomic library was replaced in plasmid pUC19 with the 321 kb fragment.
  • the bold part of the plasmid corresponds to the DNA fragment from K.marxianus, the thin part shows the part of the plasmid pUC19.
  • FIG. 6 shows the primers according to SEQ ID Nos. 4 and 5 used for the cloning of promoter and signal sequence.
  • FIG. 7 shows the signal sequence according to SEQ ID No. 3 and the associated signal peptide (with sequence for pre- and prepropeptide) of the endopolygalacturonase from Kluyveromyces marxianus.
  • a 2.198 kb Pst 1 DNA fragment containing the complete gene of endopolygalacturonase (EPG1) with the regulatory sequences was inserted into the PstI site of the multicloning site of the bacterial plasmid pUC19.
  • This construct is shown in FIG.
  • two opposite primers which had a recognition site for the restriction enzyme BstT1 at the 5 'end and had homology with the promoter, signal and / or the terminator sequence of the EPG1 gene at the 3' end, by means of the Plasmid circulating PCR a DNA fragment are generated, which could be circularized with a ligase after restriction with BspT1.
  • corresponding linkers had to be ligated in for circularization ligation or the primers contained additional sequences at the 5' end, which represented a common recognition site for the restriction enzyme.
  • defined areas of the EPG 1 region can be deleted from the plasmid by restriction and subsequent circularization.
  • the recombinant deletion plasmids were amplified in Escherichia coli and served as the basis for the expression cassettes.
  • the cassette can then be cut out via the different recognition sites for restriction enzymes in the multicloning site of the plasmid pUC19 and cloned into an episomal or integrative vector for a corresponding yeast strain.
  • the cassette can also be used directly as a PstI DNA fragment for integration into a yeast host strain.
  • the 321 bp Pvull fragment was exchanged for a Stul / Pvull DNA fragment of 2.735 kb which contains the complete endopolygalacturonase gene, including the regulatory sequences -1 142 to -1 and + 1087 to 1595 from Kluyveromyces marxianus .
  • the Stul / Pvull fragment was isolated from a recombinant Lambda GEM TM -12 bacteriophage from a genomic library of Kluyveromyce marxianus.
  • a map of the plasmid generated: “pUC19-PG1a” is shown in FIG. 5.
  • This plasmid can be used in an analogous manner to example 1 for the generation of expression cassettes. Because the cloning strategy for this plasmid of the multicloning site was completely deleted and the Stul site in the upstream promoter region of the EPG1 gene was destroyed by ligation with the Pvull site in pUC19, the Pvull site remaining downstream to the EPG1 gene and, for example, the unique Narl site in the lacZ part of the pUC19 can be used to cut out the cassette.
  • a DNA fragment can be generated which contains the promoter region and the signal sequence according to SEQ ID Nos. 1 and 3.
  • the degenerate rPEPG primer has a mismatch to the EPG1 sequence in region 1220 in the 5 'region and thereby generates a Pvull site (CAGCTG) from the EPG1 sequence: CAGTTG.
  • This point mutation leads to an amino acid exchange by changing a cysteine codon from TGT to an arginine codon after CGT.
  • this exchange is in the portion of endopolygalacturonase that is replaced by the reading frame of a foreign gene and therefore has no influence on expression.
  • the Stul / Pvull PCR Fragment can be inserted as a blunt end in the multicloning site of a corresponding vector. Via the Pvull location, open reading frames of genes can be added "in frame", the expression product of which is to be removed from the cell.
  • the expression cassette pEPG1-1 was used for the expression of the JCV VPI gene.
  • the VPIGen was PCR by using the primers:
  • JC5 (SEQ ID NO.6): 5'TCAAGCTTAAGAATGGCCCCAACAAAAAGA 3 'and
  • JC3 (SEQ ID NO.7): 5'GTAAGCTTAAGATTACAGCA I I I I I GTCTG 3 '
  • the "blunt ended" cassette was inserted into the Sma ⁇ site of the Kluyveromyces vector pKDSU.
  • the direction of transcription of the 1 P7 gene is opposite to the transcription of the. S. cerevisiae URA3 gene.
  • the recipient (BKM Y-719 ura) was transformed with the expression construct and uracil prototrophic clones were isolated.
  • the presence of the expression cassette with the reading frame of the VP1 gene was checked by means of PCR and the primers shown above.
  • Selected clones were cultivated in various liquid media.
  • the yeast cells were harvested by centrifugation and disrupted in lysis buffer (10mM TRIS-HCl, pH7.5, 0.01% TRITON X100, 1mM CaC, 100mM NaCl with 1mM PMSF) with microglass balls at 0 ° C. The glass spheres were then sedimented by centrifugation (2000 rpm) for 10 minutes.
  • the supernatant was layered on a 20% sucrose cushion (in lysis buffer) and centrifuged for 2 hours at 4 ° C and 35000rpm (Beckman L8-75 rotor SW41). The pellet was suspended in lysis buffer and layered on a CsCI gradient (1.24 to 1.38 g / ml CsCI). The centrifugation took place at 4 ° C, 35000rpm for 16 hours in the SW41 rotor.
  • "Virus like Particies" of JCV VP1 with an average Particle sizes of 45 nm were obtained from the fractions between 1.3 and 1.34 g / ml CsCI.
  • HBV s antigen Production of hepatitis BVirus surface antigen (HBV s antigen):
  • the expression cassettes pEPG1-1 and pEPG1-2 were used for the expression of the HBV s antigen gene of the main type (AYW).
  • the HBS gene was PCR-engineered using the primers:
  • HB5 (SEQ ID No. 8): 5 ⁇ GCC77A4 ⁇ ATAATGGAGAACATCACATCAGG 3 'and
  • the "blunt ended" expression cassettes were inserted into the Sma ⁇ site of the Kluyveromyces vector pKDSU.
  • the direction of transcription of the HBS gene is opposite to the transcription of the S.cerevisiae URA3 gene.
  • the recipient (BKM Y-719 ura) was transformed with the expression constructs and uracil prototrophic clones were isolated on selective medium. Total DNA was isolated from selected clones and used for the detection of the expression cassette by means of PCR. Positive clones were cultured in liquid, synthetic and in complete media and used to generate S-protein particles Yeast cultures were harvested by centrifugation and broken up in PBS buffer with I mM PMSF with microglass balls at 0 ° C.
  • the glass balls were then sedimented by centrifugation (2000 rpm) for 10 minutes.
  • the supernatant was centrifuged at 14000 rpm for 30 minutes (J20 Rotor Beckman J2-21) sedimented the yeast membrane fraction.
  • HBV s antigen sedimented with this fraction and was eluted from the fraction by 0.5% Tween-20 in PBS. After renewed centrifugation at 14000rpm, the supernatant was obtained and separated by CsCI density gradient centrifugation. Highly purified HBV s antigen particles were isolated from the 1.2g / ml CsCI fraction.
  • the yield of isolable antigen when cultivated in synthetic media was twice as high with the pEPG 1 -2 cassette than with the pEPG1 -1 cassette.

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Abstract

The invention relates to a DNA sequence which acts as a promoter in yeast cells, to an expression and optionally, secretion system containing said DNA sequence, to plasmids containing this system, to host cells that have been transformed with said DNA and to methods for producing proteins and polypeptides.

Description

REGULATIRISCHE SEQUENZEN UND EXPRESSIONKASSETTENREGULATORY SEQUENCES AND EXPRESSION CASSETTES
FÜR HEFENFOR YEARS
Die Erfindung betrifft eine DNA-Sequenz, die als Promotor in Hefezellen aktiv ist, ein dieses enthaltendes Expressions- und gegebenenfalls Sekretionssystem, dieses System enthaltende Plasmide, mit der DNA transformierte Wirtszellen sowie Verfahren zur Herstellung von Proteinen und Polypeptiden.The invention relates to a DNA sequence which is active as a promoter in yeast cells, an expression and optionally secretion system containing the same, plasmids containing this system, host cells transformed with the DNA and methods for producing proteins and polypeptides.
Die Herstellung von Peptiden und Proteinen mit gentechnischen Verfahren ist mittlerweile üblich und es stehen hierfür viele verschiedene Systeme zur Verfügung. Häufig werden Bakteriensysteme verwendet, die allerdings Nachteile haben insbesondere bei der Gewinnung von Medikamenten oder Impfstoffen, z.B. daß sie pyrogene Substanzen erzeugen, die vor der Verwendung entfernt werden müssen, oder daß sie die Polypeptide nicht glykosylieren können. Es wurden daher auch eine Reihe anderer Systeme für die Expression von Polypeptiden in eukaryontischen Zellen entwickelt. Ein hierfür weitverbreiteter Organismus ist die Hefe Saccharomyces cerevisiae, deren Genom inzwischen bekannt ist und für die Vektoren und Expressionssysteme erhältlich sind. Allerdings hat auch die Verwendung dieses Mikroorganismus Nachteile. So ist Saccharomyces zum Beispiel temperaturempfindlich, was aufwendige Einrichtungen zur Temperaturkontrolle bei der Züchtung erfordert.The production of peptides and proteins using genetic engineering is now common and there are many different systems available for this. Bacterial systems are often used, but they have disadvantages, in particular when it comes to obtaining drugs or vaccines, e.g. that they produce pyrogenic substances that must be removed before use, or that they cannot glycosylate the polypeptides. A number of other systems for the expression of polypeptides in eukaryotic cells have therefore also been developed. A widely used organism for this is the yeast Saccharomyces cerevisiae, the genome of which is now known and for which vectors and expression systems are available. However, the use of this microorganism also has disadvantages. For example, Saccharomyces is sensitive to temperature, which requires complex temperature control devices during cultivation.
Eine Gattung, die aufgrund günstiger Eigenschaften für biotechnologische Verfahren inbetracht gezogen wird, ist die Hefe Kluyveromyces. Die Arten K.lactis und K.marxianus werden als GRAS (Generally Recognized As Save) eingestuft und können daher mit derselben Sicherheit wie Saccharomyces verwendet werden. Darüberhinaus kann K.marxianus eine Vielzahl von Kohlenstoffquellen und Energiequellen für das Wachstum nutzen und ist nicht sehr temperaturempfindlich. Kluyveromyces maxrxianus kann bei Temperaturen bis zu 45 °C wachsen und läßt sich daher im Gegensatz zu den temperaturempfindlichen Saccharo- /77yce$-Stämmen einfacher züchten. Die Zellen von schnell wachsenden K.marxianυs-Stämmen können sich bei optimalen Bedingungen alle 35 Minuten teilen. Allerdings konnten diese guten Eigenschaften bisher nicht optimal ausgenutzt werden, da es für diese Hefeart an zuverlässigen, expressionsstarken variablen Promotoren mangelt und kaum Expressionssysteme zur Verfügung stehen, mit denen Proteine in guter Effektivität hergestellt werden können. Es war nun Aufgabe der vorliegenden Erfindung, einen Promotor, der für die Expression in Hefen, insbesondere Hefezellen der Gattung Kluyveromyces geeignet ist, sowie Expressionssysteme, die variabel zur Expression eingesetzt werden können, bereitzustellen.One genus that is considered due to its favorable properties for biotechnological processes is the yeast Kluyveromyces. The species K.lactis and K.marxianus are classified as GRAS (Generally Recognized As Save) and can therefore be used with the same certainty as Saccharomyces. In addition, K.marxianus can use a variety of carbon and energy sources for growth and is not very temperature sensitive. Kluyveromyces maxrxianus can grow at temperatures up to 45 ° C and is therefore easier to grow than the temperature-sensitive Saccharo / 77yce $ strains. The cells of fast growing K.marxianυs strains can divide every 35 minutes under optimal conditions. However, these good properties have so far not been able to be used optimally, since there is a lack of reliable, expressive variable promoters for this type of yeast and there are hardly any expression systems available with which proteins can be produced with good effectiveness. It was an object of the present invention to provide a promoter which is suitable for expression in yeasts, in particular yeast cells of the genus Kluyveromyces, and expression systems which can be used variably for expression.
Diese Aufgabe wird gelöst, indem erfindungsgemäß eine DNA-Sequenz, die die Nucleotidsequenz gemäß SEQ ID Nr. 1This object is achieved by a DNA sequence according to the invention which contains the nucleotide sequence according to SEQ ID No. 1
GAGGCCTGTC CGATTATTAA ACTTGCGGCA CCCGAGTTTG TGACCTTCGA CGACATGTTT TATTTCCACA CCGTAGCTAC ACTTTCTATG TAGTAAGTAG GTAGTATGGA TGGTAGCTAG TAGAAACTAA ACGAAACGAA ATAAATGTGA AATGTTAGAC GTAAAGGGGA GGGGAAGGGA AGGGGGCGGC GGAGAGACAT GCCAAGCCAT GCCATTTCAT GGCATGGCAT GTCAAGGGAT ACTGCATGCA TGCATGCATA CTTTACCAAT AGCAAAGTAA ATTGCTTTCT TCCCCCATTT GAAACTATTC CACCTCAATC CATCTTTTCT ATAATGGGTA TCACCGATCT CATGTGTTCT AATAATGCTG CAGGCAACAA CAAATCTTAA AGGCAACTTG GAATGTAATT TGGTTAATGA TAGATATCAA ACAGCAATGG TGGGCTCCAA CCGCATGGAT ATGCTCACCT TATTATCCGG AATTGTTGTT CCGCAGGAAA AAAAAAAAAC CTCGAACCAG ATATTAATTA TCCTATCATT ACTGCGTACA AAACCCGGGA ACGGTTAACC TGCAGCAGCC GTTTTGCTTA CAGTTCTCAT GCACAATCAG CCAGATTTTG CAATAGTATT AACTTAGAAT TAAGGCAACA TCTTTGGATA TGCATGTAGA GTAAGTCGTT CGAAACCATT ATTATTATTA TTATTATTAT TATTATTATT ATTATTATTA TTATTAGTAT TATTGAAATT GTTATTGTTC TTAGTTTCAC TACTATTATT ATTCATATTC ATGTTATTGA CATCGCCGAA CGACCAGCCT CCATACCGAT TAGACAGGAT CTCAAACGTG GGCTCCAGAG CTCACACATT ATGCTAAATA ACTATCTACT GTAACAGCTA CAGAAAAAAA ACTATAAAAG AGCGAGGGAT AAACCACTCT CTTGTGAATC AGGATCAGTA GGTAACTCAT AAACCTTCTT CTTTTCTCTC AAAATATCAA ATAACAGTAG TATCAACAAC GATATCGAAT AATACTAACT ACTACAACAG TAGGAACAGT AACGACAACG ACAACGATAG TAACGACAAT AACGACACCA ACAAACAACA GGAACACAGA TTAAGCTCAG AAACAAAAAA AAAAAA umfaßt, zur Verfügung gestellt wird.GAGGCCTGTC CGATTATTAA ACTTGCGGCA CCCGAGTTTG TGACCTTCGA CGACATGTTT TATTTCCACA CCGTAGCTAC ACTTTCTATG TAGTAAGTAG GTAGTATGGA TGGTAGCTAG TAGAAACTAA ACGAAACGAA ATAAATGTGA AATGTTAGAC GTAAAGGGGA GGGGAAGGGA AGGGGGCGGC GGAGAGACAT GCCAAGCCAT GCCATTTCAT GGCATGGCAT GTCAAGGGAT ACTGCATGCA TGCATGCATA CTTTACCAAT AGCAAAGTAA ATTGCTTTCT TCCCCCATTT GAAACTATTC CACCTCAATC CATCTTTTCT ATAATGGGTA TCACCGATCT CATGTGTTCT AATAATGCTG CAGGCAACAA CAAATCTTAA AGGCAACTTG GAATGTAATT TGGTTAATGA TAGATATCAA ACAGCAATGG TGGGCTCCAA CCGCATGGAT ATGCTCACCT TATTATCCGG AATTGTTGTT CCGCAGGAAA AAAAAAAAAC CTCGAACCAG ATATTAATTA TCCTATCATT ACTGCGTACA AAACCCGGGA ACGGTTAACC TGCAGCAGCC GTTTTGCTTA CAGTTCTCAT GCACAATCAG CCAGATTTTG CAATAGTATT AACTTAGAAT TAAGGCAACA TCTTTGGATA TGCATGTAGA GTAAGTCGTT CGAAACCATT ATTATTATTA TTATTATTAT TATTATTATT ATTATTATTA TTATTAGTAT TATTGAAATT GTTATTGTTC TTAGTTTCAC TACTATTATT ATTCATATTC ATGTTATTGA CATCGCCGAA CGACCAGCCT CCATACCGAT TAGACAGGAT CTCAAACGTG GGCTCCAGAG CTCACACATT ATGCTAAATA ACTATCTACT GTAACAGCTA CAGAAAAAAA ACTATAAAAG AGCGAGGGAT AAACCACTCT CTTGTGAATC AGGATCAGTA GGTAACTCAT AAACCTTCTT CTTTTCTCTC AAAATATCAA ATAACAGTAG TATCAACAAC GATATCGAAT AATACTAACT ACTACAACAG TAGGAACAGT AACGACAACACACACACACAAA
Die erfindungsgemäße Nucleotidsequenz umfaßt eine Sequenz, die als Promotor aktiv ist und liefert ein Expressionssystem, das sehr variabel ist und für Kluyveromyces geeignet ist, aber auch für andere Hefearten, insbesondere Saccharomyces cerevisiae eingesetzt werden kann.The nucleotide sequence according to the invention comprises a sequence which is active as a promoter and provides an expression system which is very variable and is suitable for Kluyveromyces, but can also be used for other types of yeast, in particular Saccharomyces cerevisiae.
Die erfindungsgemäße DNA-Sequenz gemäß SEQ ID Nr. 1 ist eine Nucleinsäure- sequenz, die regulatorische Regionen eines Gens enthält, das für das Enzym Endopolygalacturonase codiert. Das Enzym Endopolygalacturonase baut Pektin ab, indem es 1 ,4-α-D-galactosituronische Bindungen zwischen zwei nichtmethy- lierten Galacturonsäureresten spaltet. Dieses Enzym kommt unter anderem in Hefestämmen der Art Kluyveromyces marxianυs vor. Die Sequenz des Endo- polygalacturonasegens aus Kluyveromyces marxianus var. marxianus wurde in Yeast 15, 31 1 -322 (1999) veröffentlicht. Die aus der angegebenen Sequenz ableitbare Promotorregion führte jedoch nicht zuverlässig zur Expression verschiedener Proteine.The DNA sequence according to the invention according to SEQ ID No. 1 is a nucleic acid sequence which contains regulatory regions of a gene which codes for the enzyme endopolygalacturonase. The enzyme endopolygalacturonase breaks down pectin by breaking 1,4-α-D-galactosituronic bonds between two non-methylated galacturonic acid residues. This enzyme occurs in yeast strains of the species Kluyveromyces marxianυs, among others. The sequence of the endo- polygalacturonasegens from Kluyveromyces marxianus var. marxianus was published in Yeast 15, 31 1 -322 (1999). However, the promoter region that can be derived from the specified sequence did not reliably lead to the expression of various proteins.
Es wurde nun gefunden, daß eine Sequenz, die zumindest einen Teil der Nucleo- tide von SEQ ID Nr. 1 , bevorzugt mindestens die Nucleotide 1 bis 1 134 und insbesondere die gesamte Sequenz umfaßt, die Expression von Proteinen in sehr vorteilhafter und reproduzierbarer Weise ermöglicht. Die beanspruchte Sequenz gemäß SEQ ID Nr. 1 weist mehrere regulatorische Anteile auf, so daß sie unter verschiedensten Bedingungen ihre Funktion als Promotor ausüben kann.It has now been found that a sequence which comprises at least part of the nucleotides of SEQ ID No. 1, preferably at least nucleotides 1 to 1 134 and in particular the entire sequence, enables the expression of proteins in a very advantageous and reproducible manner , The claimed sequence according to SEQ ID No. 1 has several regulatory components, so that it can perform its function as a promoter under the most varied of conditions.
Der erfindungsgemäße Promotor kann durch Zugabe von Pektin zum Kulturmedium induziert werden. Dies ist vorteilhaft, da Pektin eine gut verfügbare Substanz ist und somit ein günstiges Induktionsmittel für das erfindungsgemäße System zur Verfügung steht.The promoter according to the invention can be induced by adding pectin to the culture medium. This is advantageous since pectin is a readily available substance and therefore a cheap induction agent is available for the system according to the invention.
Die beschriebenen Nucleinsäuresequenzen mit regulatorischer Aktivität schließen solche Sequenzen ein, die durch Modifizierung, Substitution, Deletion oder Insertion oder Kombinationen hiervon entstanden sind, die die gleiche oder eine bessere regulatorische Aktivität als der Promotor, der Terminator oder die Signalsequenz besitzen. Hierzu kann auch die Sequenz gemäß SEQ ID Nr. 1 mit weiteren regulatorischen Upstream-Sequenzen mit Aktivator- und/oder Repres- sorfunktionen eingeschlossen sein.The described nucleic acid sequences with regulatory activity include those sequences which have arisen by modification, substitution, deletion or insertion or combinations thereof, which have the same or a better regulatory activity than the promoter, the terminator or the signal sequence. For this purpose, the sequence according to SEQ ID No. 1 can also be included with further regulatory upstream sequences with activator and / or reporter functions.
Erfindungsgemäß werden weiterhin auch solche Sequenzen inbetracht gezogen, die mit den beanspruchten Nucleotiden oder Sequenzen eine Homologie von mindestens 80%, bevorzugter mindestens 90% und insbesondere 95% aufweisen, solange sie auch eine vergleichbare Aktivität haben.According to the invention, those sequences are furthermore considered which have a homology with the claimed nucleotides or sequences of at least 80%, more preferably at least 90% and in particular 95%, as long as they also have a comparable activity.
Bevorzugt wird die Sequenz gemäß SEQ ID Nr. 1 in Form eines Expressionssystem bzw. einer Expressionskassette für die Expression von Proteinen und Peptiden bereitgestellt. Die erfindungsgemäße Expressionskassette umfaßt in ihrer einfachsten Form eine regulatorische Sequenz, wie sie in SEQ ID Nr. 1 gezeigt ist, oder einen als Promotor aktiven Teil davon, einen Insertionsklonie- rungsort, in den das Polynucleotid für das zu exprimierende Protein einkloniert werden kann, und die Nucleotidsequenz gemäß SEQ ID Nr. 2, GCGTCTCT TTTTA I I I I I I I I I I I I I I I TTATTAACGT GAAGAAGATA AGGGAAGTCT TCAATGCGGT TCTGAATGGT TGATCCATTT CGATACCTCG GGGACTTCCT TTGAATATAT TCTGAGAGTA TGACAGTTGG TTTTCTTTCT TTCTTTCTAT TGTTTTTGTT TTTATGGAAA TATAGCTTTG ATGATTTAGG ATATTTTTTG TAGTGAACCA ATACATGCTT GATTAATATA CGTACGAGGT GGGCATTCTA CTCTCATTAT TGGTGTTTTA TTGGAGGGAA AAATTAAATC TAGGAGTATC GTTTAGAGCG CGAACGTAAT ATCCATGTTC TTCTCTTTGA AGAGGTCCCA CCATTGCTTC CCAGATAGCC AGCATTCTTC CATGATATTT TGCGCTTGTT TTGCACTGGT GACACCCTTT CGAACCAAAG ATGTCAAGTG CTGCTGATAC AACAACCTGT ATTCATACAA TTCTGGATCC ATCAGCTCAC AATCCACAGC TGAAGATACA GAAAATGATA CATGTCTCTG CAGThe sequence according to SEQ ID No. 1 is preferably provided in the form of an expression system or an expression cassette for the expression of proteins and peptides. In its simplest form, the expression cassette according to the invention comprises a regulatory sequence as shown in SEQ ID No. 1 or a part thereof active as a promoter, an insertion cloning site into which the polynucleotide for the protein to be expressed can be cloned, and the nucleotide sequence according to SEQ ID No. 2, GCGTCTCT TTTTA IIIIIIIIIIIIIII TTATTAACGT GAAGAAGATA AGGGAAGTCT TCAATGCGGT TCTGAATGGT TGATCCATTT CGATACCTCG GGGACTTCCT TTGAATATAT TCTGAGAGTA TGACAGTTGG TTTTCTTTCT TTCTTTCTAT TGTTTTTGTT TTTATGGAAA TATAGCTTTG ATGATTTAGG ATATTTTTTG TAGTGAACCA ATACATGCTT GATTAATATA CGTACGAGGT GGGCATTCTA CTCTCATTAT TGGTGTTTTA TTGGAGGGAA AAATTAAATC TAGGAGTATC GTTTAGAGCG CGAACGTAAT ATCCATGTTC TTCTCTTTGA AGAGGTCCCA CCATTGCTTC CCAGATAGCC AGCATTCTTC CATGATATTT TGCGCTTGTT TTGCACTGGT GACACCCTTT CGAACCAAAG ATGTCAAGTG CTGCTGATAC AACAACCTGT ATTCATACAA TTCTGGATCC ATCAGCTCAC AATCCACAGC TGAAGATACA GAAAATGATA CATGTCTCTG CAG
die die Terminatorsequenzregion des Endopolygalacturonasegens aus Kluyveromyces marxianus kodiert. Diese Expressionskassette kann vielfältig verwendet werden. Der Insertionsklonierungsort ist eine Schnittstelle, an der die Sequenz aufgeschnitten werden kann und das Polynucleotid für das gewünschte Protein oder Peptid einligiert werden kann. In dieser einfachsten Form wird nach Induktion bei der Expression das Protein intrazellulär produziert und nicht aus der Zelle ausgeschleust. Es kann dann nach Aufschluß der Zelle in an sich bekannter Weise gewonnen werden. Diese Ausführungsform eignet sich sowohl für kleine Peptide und Proteine, die außerhalb der Zelle instabil sind als auch für Proteine, die generell intrazellulär lokalisiert sind.which encodes the terminator sequence region of the Kluyveromyces marxianus endopolygalacturonase gene. This expression cassette can be used in many ways. The insertion cloning site is an interface where the sequence can be cut open and the polynucleotide for the desired protein or peptide can be ligated in. In its simplest form, the protein is produced intracellularly after induction during expression and is not removed from the cell. After disruption of the cell, it can then be obtained in a manner known per se. This embodiment is suitable both for small peptides and proteins that are unstable outside the cell and for proteins that are generally located intracellularly.
In einer weiteren Ausführungsform, die insbesondere für aus der Zelle auszuschleusende Proteine geeignet ist, wird ein erfindungsgemäßes Expressions- und Sekretionssystem verwendet. Dieses System umfaßt in operativer Verbindung die Nucleinsäuresequenz gemäß SEQ ID Nr. 1 oder einen als Promotor aktiven Teil davon, die Sequenz gemäß SEQ ID Nr. 2 als Terminator und, zwischen diesen beiden Sequenzen, die Signalsequenz gemäß SEQ ID Nr. 3In a further embodiment, which is particularly suitable for proteins to be removed from the cell, an expression and secretion system according to the invention is used. In operative connection, this system comprises the nucleic acid sequence according to SEQ ID No. 1 or a part thereof active as a promoter, the sequence according to SEQ ID No. 2 as a terminator and, between these two sequences, the signal sequence according to SEQ ID No. 3
ATGT TATTCAGCAA CACCTTATTG ATCGCAGCAG CTAGTGCATT ATTAGCTGAA GCTTCTCCAT TGGAAAAGAG AATGT TATTCAGCAA CACCTTATTG ATCGCAGCAG CTAGTGCATT ATTAGCTGAA GCTTCTCCAT TGGAAAAGAG A
zum Ausschleusen des Proteins. In dieser Ausführungsform erfolgt die Züchtung in an sich bekannter Weise, wobei entweder in einem kontinuierlichen Verfahren das Protein ständig ins Medium abgegeben wird und aus der Fermentationsbrühe kontinuierlich gewonnen werden kann oder in einem diskontinuierlichen Verfahren die Zellen gezüchtet, geerntet und dann das Protein aus der Brühe gewonnen werden kann. Die erfindungsgemäße Expressionskassette eignet sich sowohl zur Expression von geeigneten sich autonom replizierenden Plasmiden als auch zum Einbau in Hefechromosomen über integrative Vektoren.to remove the protein. In this embodiment, the cultivation is carried out in a manner known per se, wherein either the protein is continuously released into the medium in a continuous process and can be continuously obtained from the fermentation broth, or the cells are cultivated and harvested in a batch process and then the protein from the broth can be won. The expression cassette according to the invention is suitable both for the expression of suitable autonomously replicating plasmids and for incorporation into yeast chromosomes via integrative vectors.
Ein weiterer Gegenstand der Erfindung sind die in den Figuren 1 bis 3 näher erläuterten Plasmide pEPG1-1 , pEPG1 -2 und pEPGsec, die die erfindungsgemäßen Expressionssysteme enthalten. Diese Plasmide sind rekombinante bakterielle Plasmide und können in der vorliegenden Form zur Amplifikation der Expressionskassetten verwendet werden. Die Plasmide sind in den Mikroorganismen DSM 12919, DSM 12920, DSM 12921 oder DSM 12922*enthalten und sind mit diesen hinterlegt.The invention further relates to the plasmids pEPG1-1, pEPG1 -2 and pEPGsec, which are explained in more detail in FIGS. 1 to 3 and which contain the expression systems according to the invention. These plasmids are recombinant bacterial plasmids and can be used in the present form for the amplification of the expression cassettes. The plasmids are contained in the microorganisms DSM 12919, DSM 12920, DSM 12921 or DSM 12922 * and are deposited with them.
Es ist jedoch bevorzugt, die Plasmide, nachdem das gewünschte Polynucleotid zur Expression eines Peptids oder Proteins einligiert wurde, in E.coli zu amplifi- zieren, dann die Plasmide zu gewinnen, die Expressionskassette mit geeigneten Restriktionsendonucleasen, für die Schnittstellen an den Rändern der Expressionskassette vorgesehen sind, auszuschneiden und die Expressionskassette in einen Hefevektor einzuligieren. Die Vektoren enthalten üblicherweise Selek- tionsmarker, um erfolgreich transformierte Zellen in an sich bekannter Weise selektieren zu können.However, it is preferred to amplify the plasmids in E. coli after ligating the desired polynucleotide to express a peptide or protein, then to collect the plasmids, the expression cassette with suitable restriction endonucleases, for the interfaces at the edges of the expression cassette are intended to cut out and insert the expression cassette into a yeast vector. The vectors usually contain selection markers in order to be able to select successfully transformed cells in a manner known per se.
Die Plasmide können gegebenenfalls in f, coli vermehrt und dann in Kluyveromyces marxianus oder einen anderen Klυyveromyces-Sxamm oder auch einen anderen Hefestamm eingesetzt werden. Als Transformationssystem können beispielsweise bekannte Plasmide auf Basis des Kluyveromyces droso- pKD1 verwendet werden. Abkömmlinge dieses Plasmids eignen sich zur Verwendung in Kluyveromyces marxianus und führen bei Verwendung des erfindungsgemäßen Expressionssystems zu einer effektiven Expression und Sekretion von Fremdproteinen im entsprechenden Wirt.The plasmids can optionally be propagated in f, coli and then used in Kluyveromyces marxianus or another Klυyveromyces strain or also another yeast strain. Known plasmids based on the Kluyveromyces droso- pKD1 can be used. Descendants of this plasmid are suitable for use in Kluyveromyces marxianus and, when using the expression system according to the invention, lead to effective expression and secretion of foreign proteins in the corresponding host.
In einer anderen Ausführungsform kann aus den erfindungsgemäßen, wie oben vorbereiteten Plasmiden die Expressionskassette einschließlich des zu exprimie- renden Polynucleotids herausgeschnitten und als linearer oder zirkularisierter DNA-Strang als Integrationskassette direkt mit Hefezellen in Kontakt gebracht werden, um von diesen aufgenommen zu werden. Aufgrund der Homologie mit dem Endopolyglacturonasegen wird dann in einem Teil der behandelten Zellen die DNA in das entsprechende Chromosom durch Austausch mit dem Endo- galacturonase-Gen aufgenommen. Die Selektion erfolgreich transfizierter Hefe-In another embodiment, the expression cassette, including the polynucleotide to be expressed, can be cut out of the plasmids according to the invention and prepared as above and, as a linear or circularized DNA strand, can be brought into direct contact with yeast cells as an integration cassette in order to be taken up by them. Because of the homology with the endopolyglacturonase gene, the DNA is then taken up in part of the treated cells by exchanging it with the endogalacturonase gene. The selection of successfully transfected yeast
*Hinterlegt bei DSMZ, Mascheroder Weg l , 38124 Braunschweig zellen erfolgt bei dieser Ausführungsform über die unterschiedliche Verwertung von Pektin. * Filed at DSMZ, Mascheroder Weg l, 38124 Braunschweig In this embodiment, cells take place via the different utilization of pectin.
Die erfindungsgemäße Expressionskassette wird stabil in Chromosomen eingebaut und führt, wenn die Zellen unter optimalen Bedingungen gezüchtet werden, zu einer guten Ausbeute des gewünschten Proteins. Abhängig von der Art des zu exprimierenden Proteins oder Peptids kann die Kopienzahl des Systems eingestellt werden. Da das Endopolygalacturonasegen in dem Chromosomensatz der Hefe nur einfach vorhanden ist, wird bei einer Transfektion oder Transformation mit dem erfindungsgemäß bereitgestellten Expressionssystem auch nur pro erfolgreich transformierter Zelle eine Kopie des Expressionsvektors vorhanden sein. Falls eine höhere Kopienzahl erwünscht ist, werden in an sich bekannter Weise an die Enden der Expressionskassette Sequenzen eines in größerer Kopienzahl in dem Chromosomensatz vorliegenden Gens, z.B. für rDNA, anligiert, um eine höhere Anzahl an Austauschereignissen zu bewirken.The expression cassette according to the invention is stably built into chromosomes and, if the cells are grown under optimal conditions, leads to a good yield of the desired protein. The number of copies of the system can be adjusted depending on the type of protein or peptide to be expressed. Since the endopolygalacturonase gene is only present once in the yeast chromosome set, a transfection or transformation with the expression system provided according to the invention will also only have one copy of the expression vector per successfully transformed cell. If a higher number of copies is desired, sequences of a gene which is present in a larger number of copies in the chromosome set, e.g. for rDNA, ligated to cause a higher number of exchange events.
Im letzteren Fall wird in an sich bekannter Weise zusätzlich noch ein Marker in die Sequenz miteingebaut, damit die erfolgreich transformierten Zellen selektiert werden können. Verfahren und hierzu geeignete Marker sind dem Fachmann bekannt und bedürfen hier keiner näheren Erläuterung.In the latter case, a marker is additionally incorporated into the sequence in a manner known per se, so that the successfully transformed cells can be selected. Methods and markers suitable for this are known to the person skilled in the art and do not require any further explanation here.
Das erfindungsgemäße System ist sehr variabel. So kann z.B. nur die Sequenz gemäß SEQ ID Nr. 1 oder ein als Promotor aktiver Teil davon zusammen mit anderen Nucleinsäuresequenzen, die weitere regulatorische Sequenzen bereitstellen, und mit einer heterologen Nucleotidsequenz kombiniert werden. Es kann die Sequenz gemäß SEQ ID Nr. 1 oder ein als Promotor aktiver Teil davon mit der Sequenz von SEQ ID Nr. 2 kombiniert werden, um ein in Kluyveromyces marxianus homologes regulatorisches System bereitzustellen, in das das Polynucleotid für das zu exprimierende Protein eingesetzt wird oder aber es kann ein System aus SEQ ID Nr. 1 , SEQ ID Nr. 2 und SEQ ID Nr. 3 zusammen mit einem zu exprimierenden Gen, das ein gewünschtes Protein kodiert, kombiniert werden, um ein in die Kultur abzugebendes Produkt zu erzeugen. Da Kiυyveromyces-ZeWen mit vielen verschiedenen C-Quellen wachsen können und bezüglich weiterer Nährstoffe nicht sehr anspruchsvoll sind, darüber hinaus temperaturunempfindlich sind, wird hier ein sehr effektives System bereitgestellt. Die zuverlässige Expression der Fremdproteine wird durch die erfindungsgemäß bereitgestellte regulatorische Sequenz der Erfindung erreicht. Erfindungsgemäß wird ein System bereitgestellt, das es zuläßt, die aufgrund ihrer außergewöhnlichen physiologischen Leistungen als Wirt vielversprechende Hefeart Kluyveromyces marxianus zu nutzen.The system according to the invention is very variable. For example, only the sequence according to SEQ ID No. 1 or a part thereof active as a promoter can be combined with other nucleic acid sequences which provide further regulatory sequences and with a heterologous nucleotide sequence. The sequence according to SEQ ID No. 1 or a part thereof active as a promoter can be combined with the sequence of SEQ ID No. 2 in order to provide a regulatory system which is homologous in Kluyveromyces marxianus and in which the polynucleotide for the protein to be expressed is used or a system of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 can be combined together with a gene to be expressed, which encodes a desired protein, in order to produce a product to be released into the culture. Since Kiυyveromyces ZeWen can grow with many different C sources and are not very demanding in terms of other nutrients, and are also insensitive to temperature, a very effective system is provided here. The reliable expression of the foreign proteins is achieved by the regulatory sequence of the invention provided according to the invention. According to the invention, a system is provided which allows the promising yeast type Kluyveromyces marxianus to be used as a host due to its exceptional physiological performance.
Das erfindungsgemäße System eignet sich zur Expression von Peptiden, Poly- peptiden, Proteinen und Hybridmolekülen einschließlich glycosylierter Proteine.The system according to the invention is suitable for the expression of peptides, polypeptides, proteins and hybrid molecules including glycosylated proteins.
So kann in einer weiteren Ausführungsform die Expressionskassette auch die vollständige Sequenz des Endopolygalacturonaseenzyms oder Teile davon enthalten, wobei zwischen das Endopolygalacturonasegen und die Terminatorsequenz eine Sequenz für ein gewünschtes Protein einligiert ist. Bei dieser Ausführungsform wird dann bei der Expression ein Hybrid erhalten, von dem in an sich bekannter Weise die Endopolygalacturonase abgespalten wird.In a further embodiment, the expression cassette can also contain the complete sequence of the endopolygalacturonase enzyme or parts thereof, a sequence for a desired protein being inserted between the endopolygalacturonase gene and the terminator sequence. In this embodiment, a hybrid is then obtained during expression, from which the endopolygalacturonase is split off in a manner known per se.
Im folgenden werden einige Definitionen für Begriffe angegeben, die in der Beschreibung verwendet werden.Some definitions of terms used in the description are given below.
Danach ist ein "Expressionsvektor" ein DNA-Molekül, das linear oder ringförmig sein kann und ein Segment enthält, das eine Sequenz für ein interessierendes Protein oder Peptid codiert, das operativ verbunden ist mit regulatorischen Sequenzen. Diese regulatorischen Sequenzen schließen mindestens Promotor- und Terminatorsequenzen ein. Der Expressionsvektor kann zusätzlich selektierbare Marker und weitere regulatorische Elemente enthalten und muß die Übertragung und Vermehrung in Wirtszellen ermöglichen. Die Replikation der Expressionsvektoren kann autonom oder durch Integration in das Wirtsgenom erfolgen.Thereafter, an "expression vector" is a DNA molecule that can be linear or circular and contains a segment that encodes a sequence for a protein or peptide of interest that is operatively linked to regulatory sequences. These regulatory sequences include at least promoter and terminator sequences. The expression vector can additionally contain selectable markers and other regulatory elements and must enable the transfer and multiplication in host cells. The expression vectors can be replicated autonomously or by integration into the host genome.
Der Ausdruck "DNA" oder "Polynucleotid" schließt polymere Formen von Desoxyribonucieotiden und Ribonucleotiden beliebiger Länge und beliebiger Modifikation in einzel- und doppelsträπgiger Form ein.The term "DNA" or "polynucleotide" includes polymeric forms of deoxyribonucieotides and ribonucleotides of any length and any modification in single and double-stranded form.
Der Ausdruck "operativ verbunden" bedeutet, daß die einzelnen Segmente so angeordnet sind, daß sie dem vorgesehenen Zweck dienen, d.h. die Transkription initiieren können und die Expression fördern können von dem Replikations- startpunkt bis zur Terminationssequenz.The term "operatively connected" means that the individual segments are arranged so that they serve the intended purpose, i.e. can initiate transcription and can promote expression from the start of replication to the termination sequence.
Die beanspruchten Sequenzen können weitere kurze Sequenzen aufweisen, die die biologische Aktivität des Moleküls nicht stören. Weiterhin umfassen die beanspruchten Sequenzen auch allelische Varianten der Sequenz, d.h. alternative Formen des Gens, die durch Mutation entstanden sind. Der Begriff "Protein oder Peptid" bezieht sich auf eine molekulare Kette von Aminosäuren mit biologischer Aktivität. Die Proteine und/oder Polypeptide können in vivo oder in vitro modifiziert werden, z.B. durch Glycosylierung und Phosphorylierung. Unter Hybridmolekülen werden Moleküle verstanden, die sowohl homologe Teile als auch heterologe Teile umfassen, z.B. solche Proteine, die eine Kombination aus Endopolygalacturonase oder Teilen davon mit einem Fremdprotein umfassen, oder z.B. Nucleotidsequenzen, bei denen DNA aus Kluyveromyces marxianus mit DNA aus anderen Mikroorganismen kombiniert ist.The claimed sequences can have further short sequences that do not interfere with the biological activity of the molecule. Furthermore, the claimed sequences also include allelic variants of the sequence, ie alternative forms of the gene which have arisen from mutation. The term "protein or peptide" refers to a molecular chain of amino acids with biological activity. The proteins and / or polypeptides can be modified in vivo or in vitro, for example by glycosylation and phosphorylation. Hybrid molecules are understood to mean molecules which comprise both homologous parts and heterologous parts, for example those proteins which comprise a combination of endopolygalacturonase or parts thereof with a foreign protein, or for example nucleotide sequences in which DNA from Kluyveromyces marxianus is combined with DNA from other microorganisms ,
Die erfindungsgemäße Expressionskassette ist unter anderem für Hefen der Stämme Kluyveromyces und Saccharomyces geeignet und wird bevorzugt in Hefestämmen der Art Kluyveromyces marxianus var. marxianus verwendet. Ein besonders bevorzugter Stamm mit besonders günstigen Expressionseigenschaften ist Kluyveromyces marxianus var. marxianus BKM Y-719, der bei Siekstele et al. 1999 (Yeast 1 5, 31 1 bis 322 (1999)) beschrieben wurde.The expression cassette according to the invention is suitable, inter alia, for yeasts of the strains Kluyveromyces and Saccharomyces and is preferably used in yeast strains of the species Kluyveromyces marxianus var. Marxianus. A particularly preferred strain with particularly favorable expression properties is Kluyveromyces marxianus var. Marxianus BKM Y-719, which was described by Siekstele et al. 1999 (Yeast 1 5, 31 1 to 322 (1999)).
Ein weiterer Gegenstand der Erfindung ist ein Verfahren zur Herstellung eines rekombinanten Proteins, das dadurch gekennzeichnet ist, daß man eine Hefezelle mit einem sich autonom replizierenden Plasmid, das eine erfindungsgemäße Expressionskassette und ein Polynucleotid, das ein Fremdprotein kodiert, umfaßt, transfiziert oder transformiert, die Hefezelle unter Bedingungen, die zur Expression des Fremdproteins geeignet sind, züchtet und das Protein gewinnt.Another object of the invention is a process for the production of a recombinant protein, which is characterized in that a yeast cell is transfected or transformed with an autonomously replicating plasmid which comprises an expression cassette according to the invention and a polynucleotide which encodes a foreign protein Yeast cell is grown under conditions suitable for the expression of the foreign protein and the protein wins.
Gegenstand der Erfindung ist auch ein Verfahren zur Herstellung eines rekombinanten Proteins, das dadurch gekennzeichnet ist, daß man eine erfindungsgemäße Expressionskassette in eine Hefezelle bringt, wo die Expressionskassette in ein Chromosom eingebaut wird, die Zelle züchtet und anschließend das Protein gewinnt. Besonders bevorzugt wird die erfindungsgemäße Expressionskassette als Modul eingesetzt das die Konstruktion von episomalen oder integra- tiven Expressionsvektoren, die die regulatorischen Sequenzen gemäß SEQ ID Nr. 1 , 2 und/oder 3 enthalten, ermöglicht.The invention also relates to a method for producing a recombinant protein, which is characterized in that an expression cassette according to the invention is placed in a yeast cell, where the expression cassette is built into a chromosome, the cell is grown and then the protein is obtained. The expression cassette according to the invention is particularly preferably used as a module which enables the construction of episomal or integrative expression vectors which contain the regulatory sequences according to SEQ ID No. 1, 2 and / or 3.
Das erfindungsgemäße Expressionssystem ist geeignet zur Expression verschiedener heterologer Proteine. Besonders bevorzugt wird das System verwendet zur Expression von HBVS-Antigen (Hepatitis-B-Virus, Surface-Antigen) und Virusprotein 1 aus Polyoma-Virus. Diese Proteine sind antigene Proteine und können besonders vorteilhaft als Impfstoffe eingesetzt werden. Die Erfindung wird durch die beigefügten Figuren und die folgenden Beispiele erläutert.The expression system according to the invention is suitable for the expression of various heterologous proteins. The system is particularly preferably used for the expression of HBVS antigen (hepatitis B virus, surface antigen) and virus protein 1 from polyoma virus. These proteins are antigenic proteins and can be used particularly advantageously as vaccines. The invention is illustrated by the accompanying figures and the following examples.
Die Figuren 1 bis 4 zeigen Plasmide mit den erfindungsgemäßen Expressionskassetten, Figur 6 zeigt die in Beispiel 2 verwendeten Primer.Figures 1 to 4 show plasmids with the expression cassettes according to the invention, Figure 6 shows the primers used in Example 2.
Fig. 1 zeigt das Plasmid pEPG1-1 . Dieses Plasmid enthält eine Expressionskassette mit dem erfindungsgemäßen Promotor gemäß SEQ ID Nr. 1 (als EPGI prom bezeichnet), einem Insertionsklonierungsort, der mit BspT1 geschnitten werden kann, und der erfindungsgemäßen Terminatorsequenz gemäß SEQ ID Nr.2 (als EPGIterm bezeichnet). Diese Expressionskassette wurde in den Multicloning Site des Plasmids pUC19 einligiert. Der Insertionsklonierungsort, der mit BspT1 geschnitten werden kann, ersetzt den offenen Leserahmen des Endopolygalacturonasegens, das entfernt wurde.1 shows the plasmid pEPG1-1. This plasmid contains an expression cassette with the promoter according to the invention according to SEQ ID No. 1 (referred to as EPGI prom), an insertion cloning site which can be cut with BspT1, and the terminator sequence according to SEQ ID No. 2 (referred to as EPGIterm). This expression cassette was ligated into the multicloning site of the plasmid pUC19. The insertion cloning site that can be cut with BspT1 replaces the open reading frame of the endopolygalacturonase gene that was removed.
Fig. 2 zeigt das Plasmid pEPG1-2. Dieses Plasmid enthält die erfindungsgemäße Expressionskassette, die eine Sequenz gemäß SEQ ID Nr. 1 mit den Nucleotiden 572 bis 1 134 (mit EPGI prom bezeichnet), die als Promotor aktiv ist, einen Insertionsklonierungsort, der mit BspT1 geschnitten werden kann, und eine Terminatorsequenz, die die Nucleotide 28 bis 541 von SEQ ID Nr. 2 (als EPGIterm bezeichnet) umfaßt. Der Insertionsklonierungsort ersetzt den offenen Leserahmen des Endopolygalacturonasegens einschließlich der Sequenzen -1 bis -12 und 1087 bis 1 1 15 dieses Gens.Fig. 2 shows the plasmid pEPG1-2. This plasmid contains the expression cassette according to the invention, which has a sequence according to SEQ ID No. 1 with nucleotides 572 to 1 134 (designated EPGI prom), which is active as a promoter, an insertion cloning site which can be cut with BspT1, and a terminator sequence, which comprises nucleotides 28 to 541 of SEQ ID No. 2 (referred to as EPGIterm). The insertion cloning site replaces the open reading frame of the endopolygalacturonase gene including the sequences -1 to -12 and 1087 to 1115 of this gene.
Fig. 3 zeigt das Plasmid pEPGsec. Dieses Plasmid enthält ein Expressions- und Sekretionssystem der Erfindung. Das Plasmid enthält eine Expressionskassette mit einem Promotor gemäß SEQ ID Nr. 1 (als EPGI prom bezeichnet), einem Terminator gemäß SEQ ID Nr. 2 (als EPGIterm bezeichnet), einer Signalsequenz gemäß SEQ ID Nr. 3 (als EOGI Iyd bezeichnet) und zwei Schnittstellen, um die gewünschte Nucleinsäuresequenz für das zu exprimierende Protein einzusetzen. Diese Expressionskassette wurde in den Multicloning Site des Plasmids pUC19 einligiert. Der offene Leserahmen des Endopolygalacturonasegens wurde von der Position 75 bis 1084 entfernt und durch einen Linker mit den Klonierungsorten Eco 1471 und BpU 1 1021 ersetzt.Fig. 3 shows the plasmid pEPGsec. This plasmid contains an expression and secretion system of the invention. The plasmid contains an expression cassette with a promoter according to SEQ ID No. 1 (referred to as EPGI prom), a terminator according to SEQ ID No. 2 (referred to as EPGIterm), a signal sequence according to SEQ ID No. 3 (referred to as EOGI Iyd) and two interfaces to insert the desired nucleic acid sequence for the protein to be expressed. This expression cassette was ligated into the multicloning site of the plasmid pUC19. The open reading frame of the endopolygalacturonase gene was removed from positions 75 to 1084 and replaced by a linker with the cloning sites Eco 1471 and BpU 1 1021.
Figur 4 zeigt das Plasmid pUC19PG. Ein 2,198 kb Pstl-DNA-Restriktions- fragment aus einem rekombinanten LambdaGEM™-12-Bakterioρhagen einer genomischen Genbank von Kluyveromyces marxianus wurde in den Pstl-Restrik- tionsort des Multicloning Site des Plasmids pUC19 ligiert. Fig. 5 zeigt das Plasmid pUC19-PG1 a. Ein 2,735 kb großes Stul-Pvull-DNA- Restriktionsfragment aus einem rekombinanten LambdaGEM™-12-Bakteriophagen einer genomischen Genbank von Kluyveromyces marxianus wurde im Plasmid pUC19 gegen das 321 kb große Fragment ausgetauscht. Der fett gedruckte Teil des Plasmids entspricht dem DNA-Fragment aus K.marxianus, der dünn gedruckte Teil stellt den Anteil des Plasmids pUC19 dar.Figure 4 shows the plasmid pUC19PG. A 2.198 kb PstI DNA restriction fragment from a recombinant LambdaGEM ™ 12 bacteriophage from a Kluyveromyces marxianus genomic library was ligated into the PstI restriction site of the multicloning site of the plasmid pUC19. 5 shows the plasmid pUC19-PG1 a. A 2.735 kb Stul-Pvull DNA restriction fragment from a recombinant LambdaGEM ™ 12 bacteriophage from a Kluyveromyces marxianus genomic library was replaced in plasmid pUC19 with the 321 kb fragment. The bold part of the plasmid corresponds to the DNA fragment from K.marxianus, the thin part shows the part of the plasmid pUC19.
Figur 6 zeigt die für die Klonierung von Promotor und Signalsequenz verwendeten Primer gemäß SEQ ID Nr. 4 und 5.FIG. 6 shows the primers according to SEQ ID Nos. 4 and 5 used for the cloning of promoter and signal sequence.
Figru 7 zeigt die Signalsequenz gemäß SEQ ID Nr. 3 und das zugehörige Signalpeptid (mit Sequenz für Pre- und Prepropeptid) der Endopolygalacturonase aus Kluyveromyces marxianus.FIG. 7 shows the signal sequence according to SEQ ID No. 3 and the associated signal peptide (with sequence for pre- and prepropeptide) of the endopolygalacturonase from Kluyveromyces marxianus.
Bei der DSMZ wurden 4 Mikroorganismen gemäß den Anforderungen des Budapester Vertrags hinterlegt, die die Plasmide gemäß Fig. 1 , Fig. 2, Fig. 3 und Fig. 4 enthalten. Es sind dies £. coli pEPG1-1 , hinterlegt unter der Hinterlegungsnummer DSM 12919, E. coli pUC19PG, hinterlegt DSM 12920, E. coli pEPGseq, hinterlegt mit der Nummer DSM 12921 und £. coli pEPG1-2 mit der Hinterlegungsnummer DSM 12922.Four microorganisms were stored at the DSMZ in accordance with the requirements of the Budapest Treaty, which contain the plasmids according to FIG. 1, 2, 3 and 4. This is £. coli pEPG1-1, deposited under the deposit number DSM 12919, E. coli pUC19PG, deposited DSM 12920, E. coli pEPGseq, deposited with the numbers DSM 12921 and £. coli pEPG1-2 with the accession number DSM 12922.
Beispiel 1example 1
Herstellung des Grundplasmids zur Erzeugung der ExpressionskassettenProduction of the basic plasmid for the generation of the expression cassettes
Ein Pst 1 -DNA-Fragment mit 2,198 kb, das das komplette Gen der Endopolygalacturonase (EPG1 ) mit den regulatorischen Sequenzen enthält, wurde in die Pstl-Schnittstelle des Multicloning Site des bakteriellen Plasmides pUC19 inseriert. In Fig. 4 ist dieses Konstrukt dargestellt. Unter Verwendung von jeweils zwei entgegengesetzten Primern, die am 5 '-Ende einen Erkennungsort für das Restriktionsenzym BstT1 trugen und am 3'-Ende Homologie mit der Promotor-, Signal- und/oder der Terminatorsequenz des EPG1-Gens aufwiesen, konnte mittels um das Plasmid umlaufender PCR ein DNA-Fragment erzeugt werden, das nach Restriktion mit BspT1 mit einer Ligase zirkularisiert werden konnte. Bei Verwendung verschiedener Erkennungsorte für Restriktionsenzyme an den 5'- Enden der PCR-Primer mußten zur Zirkularisationsligation entsprechende Linker einligiert werden oder die Primer enthielten zusätzliche Sequenzen am 5 '-Ende, die einen gemeinsamen Erkennungsort für das Restriktionsenzym darstellten. Je nach Auswahl der Primer können definierte Bereiche der EPG 1 -Region durch Restriktion und anschließende Zirkularisierung aus dem Plasmid deletiert werden. Die rekombinanten Deletionsplasmide wurden in Escherichia coli amplifiziert und dienten als Grundlage für die Expressionskassetten. Über die verschiedenen Erkennungsstellen für Restriktionsenzyme im Multicloning Site des Plasmids pUC19 kann dann die Kassette ausgeschnitten und in einen episomalen oder integrativen Vektor für einen entsprechenden Hefestamm kloniert werden. Die Kassette kann auch direkt als Pstl-DNA-Fragment zur Integration in einen Hefe- Wirtsstamm verwendet werden.A 2.198 kb Pst 1 DNA fragment containing the complete gene of endopolygalacturonase (EPG1) with the regulatory sequences was inserted into the PstI site of the multicloning site of the bacterial plasmid pUC19. This construct is shown in FIG. Using in each case two opposite primers, which had a recognition site for the restriction enzyme BstT1 at the 5 'end and had homology with the promoter, signal and / or the terminator sequence of the EPG1 gene at the 3' end, by means of the Plasmid circulating PCR a DNA fragment are generated, which could be circularized with a ligase after restriction with BspT1. When using different recognition sites for restriction enzymes at the 5 'ends of the PCR primers, corresponding linkers had to be ligated in for circularization ligation or the primers contained additional sequences at the 5' end, which represented a common recognition site for the restriction enzyme. Depending on the choice of primer, defined areas of the EPG 1 region can be deleted from the plasmid by restriction and subsequent circularization. The recombinant deletion plasmids were amplified in Escherichia coli and served as the basis for the expression cassettes. The cassette can then be cut out via the different recognition sites for restriction enzymes in the multicloning site of the plasmid pUC19 and cloned into an episomal or integrative vector for a corresponding yeast strain. The cassette can also be used directly as a PstI DNA fragment for integration into a yeast host strain.
Beispiel 2Example 2
Herstellung eines rekombinanten Plasmides mit Sequenz ID Nr.1 , Nr.2 und Nr.3Production of a recombinant plasmid with sequence ID No. 1, No. 2 and No. 3
Im bakteriellen Grundplasmid pUC19 wurde das 321 bp lange Pvull Fragment gegen ein Stul/Pvull DNA Fragment von 2,735 kb, das das komplette Endopoly- galacturonasegen, einschließlich der regulatorischen Sequenzen -1 142 bis -1 und + 1087 bis 1595 aus Kluyveromyces marxianus enthält, ausgetauscht. Das Stul/Pvull Fragment wurde dazu aus einem rekombinanten Lambda GEM™-12 Bakteriophagen einer genomischen Genbank von Kluyveromyce marxianus isoliert. Eine Karte des erzeugten Plasmides: „pUC19-PG1a" ist in Figur Nr. 5 dargestellt. Dieses Plasmid kann in analoger Weise zum Beispiel 1 für die Erzeugung von Expressionskassetten verwendet werden. Da durch die Klonierungsstrategie bei diesem Plasmid der Multicloning Site komplett deletiert wurde und der Stul Ort im Upstream-Promotorbereich des EPG1 Gens durch Ligation mit dem Pvull Ort im pUC19 zerstört wurde, können zum Ausschneiden der Kassette der downstream zum EPG1 Gen verbliebene Pvull Ort und z.B. der unikale Narl Ort im lacZ-Teil des pUC19 verwendet werden.In the basic bacterial plasmid pUC19, the 321 bp Pvull fragment was exchanged for a Stul / Pvull DNA fragment of 2.735 kb which contains the complete endopolygalacturonase gene, including the regulatory sequences -1 142 to -1 and + 1087 to 1595 from Kluyveromyces marxianus , The Stul / Pvull fragment was isolated from a recombinant Lambda GEM ™ -12 bacteriophage from a genomic library of Kluyveromyce marxianus. A map of the plasmid generated: “pUC19-PG1a” is shown in FIG. 5. This plasmid can be used in an analogous manner to example 1 for the generation of expression cassettes. Because the cloning strategy for this plasmid of the multicloning site was completely deleted and the Stul site in the upstream promoter region of the EPG1 gene was destroyed by ligation with the Pvull site in pUC19, the Pvull site remaining downstream to the EPG1 gene and, for example, the unique Narl site in the lacZ part of the pUC19 can be used to cut out the cassette.
Mit Hilfe der PCR und unter Verwendung der in Figur Nr.6 dargestellten Primer (SEQ ID Nr. 4 und 5) kann ein DNA Fragment generiert werden, das die Promotorregion und die Signalsequenz nach SEQ ID Nr.1 und Nr.3 enthält. Der degenerierte rPEPG Primer hat im 5' Bereich ein Mismatch zur EPG1 Sequenz im Bereich 1220 und generiert dadurch aus der EPG1 Sequenz: CAGTTG einen Pvull Ort (CAGCTG). Diese Punktmutation (Transition) führt zu einem Aminosäureaustausch, indem ein Cystein-Codon von TGT in ein Arginin-Codon nach CGT verändert wird. Dieser Austausch befindet sich aber im Anteil der Endopolygalacturonase, der durch den Leserahmen eines Fremdgenes ersetzt wird und hat somit keinen Einfluß auf die Expression. Das Stul/Pvull PCR- Fragment kann als blunt End in den Multicloning Site eines entsprechenden Vectors inseriert werden. Über den Pvull Ort können „in Frame" offene Leserahmen von Genen angefügt werden, deren Expressionsprodukt aus der Zelle ausgeschleust werden soll.Using the PCR and using the primers shown in FIG. 6 (SEQ ID Nos. 4 and 5), a DNA fragment can be generated which contains the promoter region and the signal sequence according to SEQ ID Nos. 1 and 3. The degenerate rPEPG primer has a mismatch to the EPG1 sequence in region 1220 in the 5 'region and thereby generates a Pvull site (CAGCTG) from the EPG1 sequence: CAGTTG. This point mutation (transition) leads to an amino acid exchange by changing a cysteine codon from TGT to an arginine codon after CGT. However, this exchange is in the portion of endopolygalacturonase that is replaced by the reading frame of a foreign gene and therefore has no influence on expression. The Stul / Pvull PCR Fragment can be inserted as a blunt end in the multicloning site of a corresponding vector. Via the Pvull location, open reading frames of genes can be added "in frame", the expression product of which is to be removed from the cell.
Beispiel 3Example 3
Herstellung von Polyoma JCV MAJOR COAT PROTEIN VP1 :Production of Polyoma JCV MAJOR COAT PROTEIN VP1:
Für die Expression des JCV VPI-Gens wurde die Expressionskassette pEPG1 -1 verwendet. Das VPIGen wurde mittels PCR unter Verwendung der Primer:The expression cassette pEPG1-1 was used for the expression of the JCV VPI gene. The VPIGen was PCR by using the primers:
JC5 (SEQ ID NR.6): 5'TCAAGCTTAAGAATGGCCCCAACAAAAAGA 3' undJC5 (SEQ ID NO.6): 5'TCAAGCTTAAGAATGGCCCCAACAAAAAGA 3 'and
JC3 (SEQ ID NR. 7): 5'GTAAGCTTAAGATTACAGCA I I I I I GTCTG 3'JC3 (SEQ ID NO.7): 5'GTAAGCTTAAGATTACAGCA I I I I I GTCTG 3 '
amplifiziert und als Bspl\ Fragment in die Expressionskassette von pEPG1-1 ein- kloniert. Die Kassette wurde mit PsA aufgespalten und die DNA-Fragmente an ihren Enden mittels T4-Polymerase zu stumpfen Enden umgebaut.amplified and cloned as a Bspl \ fragment into the expression cassette of pEPG1-1. The cassette was split with PsA and the ends of the DNA fragments were converted to blunt ends using T4 polymerase.
Die „blunt ended" Kassette wurden in den Sma\ Ort des Kluyveromyces Vektors pKDSU inseriert. Die Transkriptionsrichtung des 1 P7-Gens ist dabei entgegengesetzt zur Transkription des. S.cerevisiae URA3 Gens.The "blunt ended" cassette was inserted into the Sma \ site of the Kluyveromyces vector pKDSU. The direction of transcription of the 1 P7 gene is opposite to the transcription of the. S. cerevisiae URA3 gene.
Der Rezipient (BKM Y-719 ura) wurde mit dem Expressionskonstrukt transformiert und Uracil prototrophe Klone wurden isoliert. Das Vorhandensein der Expressionskassette mit dem Leserahmen des VP1 Gens wurde mittels PCR und den oben gezeigten Primern überprüft. Ausgewählte Klone wurden in verschiedenen flüssigen Medien kultiviert. Die Hefezellen wurden durch Zentrifugation geerntet und in Lysis Puffer (10mM TRIS-HCI, pH7.5, 0,01 %TRITON X100, 1 mM CaC , 100mM NaCI mit 1 mM PMSF) mit Mikroglaskugeln bei 0°C aufgebrochen . Die Glaskugeln wurden anschließend durch Zentrifugation (2000 rpm) für 10 Minuten sedimentiert. Der Überstand wurde auf ein 20%iges Saccharosekissen (in Lysispuffer) geschichtet und 2 Stunden bei 4°C und 35000rpm (Beckman L8-75 rotor SW41 ) zentrifugiert. Das Pellet wurde in Lysispuffer suspendiert und auf einen CsCI-Gradienten (1 ,24 bis 1 ,38 g/ml CsCI) geschichtet. Die Zentrifugation erfolgte bei 4°C, 35000rpm für 16 Stunden im SW41 Rotor. „Virus like Particies" des JCV VP1 mit einer durchschnittlichen Partikelgröße von 45nm wurden aus den Fraktionen zwischen 1 ,3 und 1 ,34g/ml CsCI gewonnen.The recipient (BKM Y-719 ura) was transformed with the expression construct and uracil prototrophic clones were isolated. The presence of the expression cassette with the reading frame of the VP1 gene was checked by means of PCR and the primers shown above. Selected clones were cultivated in various liquid media. The yeast cells were harvested by centrifugation and disrupted in lysis buffer (10mM TRIS-HCl, pH7.5, 0.01% TRITON X100, 1mM CaC, 100mM NaCl with 1mM PMSF) with microglass balls at 0 ° C. The glass spheres were then sedimented by centrifugation (2000 rpm) for 10 minutes. The supernatant was layered on a 20% sucrose cushion (in lysis buffer) and centrifuged for 2 hours at 4 ° C and 35000rpm (Beckman L8-75 rotor SW41). The pellet was suspended in lysis buffer and layered on a CsCI gradient (1.24 to 1.38 g / ml CsCI). The centrifugation took place at 4 ° C, 35000rpm for 16 hours in the SW41 rotor. "Virus like Particies" of JCV VP1 with an average Particle sizes of 45 nm were obtained from the fractions between 1.3 and 1.34 g / ml CsCI.
Beispiel 4Example 4
Herstellung von Hepatitis BVirus Surface-Antigen (HBV s-Antigen):Production of hepatitis BVirus surface antigen (HBV s antigen):
Für die Expression des HBV s-Antigen Gens des Suptyps (AYW) wurden die Expressionskassetten pEPG1-1 und pEPG1-2 verwendet. Das HBS Gen wurde mittels PCR unter Verwendung der Primer:The expression cassettes pEPG1-1 and pEPG1-2 were used for the expression of the HBV s antigen gene of the main type (AYW). The HBS gene was PCR-engineered using the primers:
HB5 (SEQ ID Nr. 8) : 5ΑGCC77A4σATAATGGAGAACATCACATCAGG 3' undHB5 (SEQ ID No. 8): 5ΑGCC77A4σATAATGGAGAACATCACATCAGG 3 'and
HB3 SEQ ID Nr. 9): 5'TGAC7 4^GTTAAATGTATACCCAAAG 3'HB3 SEQ ID No. 9): 5'TGAC7 4 ^ GTTAAATGTATACCCAAAG 3 '
amplifiziert und als Bspl\ Fragment in die Expressionskassetten einkloniert. Die Kassetten wurden mit Bam \ aufgespalten und die DNA-Fragmente an ihren Enden mittels Klenow-Polymerase zu stumpfen Enden aufgefüllt.amplified and cloned as Bspl \ fragment in the expression cassettes. The cassettes were cleaved with Bam and the ends of the DNA fragments filled in to blunt ends using Klenow polymerase.
Die „blunt ended" Expressionskassetten wurden in den Sma\ Ort des Kluyveromyces Vektors pKDSU inseriert. Die Transkriptionsrichtung des HBS- Gens ist dabei entgegengesetzt zur Transkription des S.cerevisiae URA3 Gens. Der Rezipient (BKM Y-719 ura) wurde mit den Expressionskonstrukten transformiert und Uracil prototrophe Klone wurden auf Selektivmedium isoliert. Von ausgewählten Klonen wurde Gesamt-DNA isoliert und diese zum Nachweis der Expressionskassette mittels PCR eingesetzt. Positive Klone wurden in flüssigen, synthetischen und in Komplettmedien kultiviert und zur Erzeugung von S-Pro- teinpartikeln eingesetzt. Entsprechende Hefekulturen wurden durch Zentrifugation geerntet und in PBS Puffer mit I mM PMSFmit Mikroglaskugeln bei 0°C aufgebrochen. Die Glaskugeln wurden anschließend durch Zentrifugation (2000 rpm) für 10 Minuten sedimentiert. Aus dem Überstand wurde durch 30 minütige Zentrifugation bei 14000 rpm (J20 Rotor Beckman J2-21 ) die Hefemembranfraktion sedimentiert. HBV s-Antigen sedimentierte mit dieser Fraktion und wurde durch 0,5% Tween-20 in PBS aus der Fraktion eluiert. Nach erneuter Zentrifugation bei 14000rpm wurde der Überstand gewonnen und durch CsCI- Dichtegradienten-Zentrifugation aufgetrennt. Hochgereinigte HBV s-Antigenpar- tikel wurden aus der 1.2g/ml CsCI Fraktion isoliert.The "blunt ended" expression cassettes were inserted into the Sma \ site of the Kluyveromyces vector pKDSU. The direction of transcription of the HBS gene is opposite to the transcription of the S.cerevisiae URA3 gene. The recipient (BKM Y-719 ura) was transformed with the expression constructs and uracil prototrophic clones were isolated on selective medium. Total DNA was isolated from selected clones and used for the detection of the expression cassette by means of PCR. Positive clones were cultured in liquid, synthetic and in complete media and used to generate S-protein particles Yeast cultures were harvested by centrifugation and broken up in PBS buffer with I mM PMSF with microglass balls at 0 ° C. The glass balls were then sedimented by centrifugation (2000 rpm) for 10 minutes. The supernatant was centrifuged at 14000 rpm for 30 minutes (J20 Rotor Beckman J2-21) sedimented the yeast membrane fraction. HBV s antigen sedimented with this fraction and was eluted from the fraction by 0.5% Tween-20 in PBS. After renewed centrifugation at 14000rpm, the supernatant was obtained and separated by CsCI density gradient centrifugation. Highly purified HBV s antigen particles were isolated from the 1.2g / ml CsCI fraction.
Die Ausbeute an isolierbarem Antigen war bei Kultivierung in synthetischen Medien mit der pEPG 1 -2 Kassette zweifach höher als mit der pEPG1 -1 Kassette. The yield of isolable antigen when cultivated in synthetic media was twice as high with the pEPG 1 -2 cassette than with the pEPG1 -1 cassette.

Claims

Patentansprüche claims
1. DNA-Sequenz umfassend die Nucleotidsequenz von SEQ ID Nr. 1.1. DNA sequence comprising the nucleotide sequence of SEQ ID No. 1.
2. Hefeexpressionssystem enthaltend in operativer Verbindung die Nucleotidsequenz von SEQ ID Nr. 1 oder einen als Promotor aktiven Teil davon, eine Insertionsklonierungsstelle und die Nucleotidsequenz von SEQ ID Nr. 2 oder einen als Terminator aktiven Teil davon.2. Yeast expression system containing, in operative connection, the nucleotide sequence of SEQ ID No. 1 or a part thereof active as a promoter, an insertion cloning site and the nucleotide sequence of SEQ ID No. 2 or a part thereof active as a terminator.
3. Hefeexpressions- und Sekretionssystem umfassend in operativer Verbindung eine Sequenz gemäß SEQ ID Nr. 1 oder einen als Promotor aktiven Teil davon, die Nucleotidsequenz von SEQ ID Nr. 3, eine Insertionsklonierungsstelle und die Nucleotidsequenz von SEQ ID Nr. 2 oder einen als Terminator aktiven Teil davon.3. Yeast expression and secretion system comprising, in operative connection, a sequence according to SEQ ID No. 1 or a part thereof active as a promoter, the nucleotide sequence of SEQ ID No. 3, an insertion cloning site and the nucleotide sequence of SEQ ID No. 2 or one as a terminator active part of it.
4. Plasmid pEPG1-1 enthaltend eine Hefeexpressionskassette gemäß Anspruch 2 hinterlegt mit der Hinterlegungsnr. DSM 12919.4. Plasmid pEPG1-1 containing a yeast expression cassette according to claim 2 deposited with the deposit no. DSM 12919.
5. Plasmid pEPG1 -2 enthaltend eine Hefeexpressionskassette gemäß Anspruch 2 hinterlegt mit der Hinterlegungsnr. DSM 12922.5. plasmid pEPG1 -2 containing a yeast expression cassette according to claim 2 deposited with the deposit no. DSM 12922.
6. Plasmid pUC19PG hinterlegt mit der Hinterlegungsnr. 12920.6. Plasmid pUC19PG deposited with the deposit no. 12,920th
7. Plasmid pEPGsec enthaltend eine Hefeexpressionskassette gemäß Anspruch 3 hinterlegt mit der Hinterlegungsnr. DSM 12921 .7. plasmid pEPGsec containing a yeast expression cassette according to claim 3 deposited with the deposit no. DSM 12921.
8. Expressionsvektor enthaltend in operativer Verbindung einen Promotor mit der Sequenz von SEQ ID N1 r. 1 oder einen als Promotor aktiven Teil davon, ein Polynucleotid, das ein Fremdprotein kodiert, und eine Terminatorsequenz.8. Expression vector containing, in operative connection, a promoter with the sequence of SEQ ID N1 r. 1 or a portion thereof active as a promoter, a polynucleotide encoding a foreign protein, and a terminator sequence.
9. Expressionsvektor nach Anspruch 8, der zusätzlich noch eine Signalsequenz zwischen Promotor und Polynucleotid enthält.9. Expression vector according to claim 8, which additionally contains a signal sequence between promoter and polynucleotide.
10. Expressionsvektor nach Anspruch 9, dadurch gekennzeichnet, daß die Signalsequenz eine Sequenz gemäß SEQ ID Nr. 3 ist. 10. Expression vector according to claim 9, characterized in that the signal sequence is a sequence according to SEQ ID No. 3.
1 1 . Expressionsvektor nach einem der Ansprüche 8 bis 10, dadurch gekennzeichnet, daß das Polynucleotid ein antigenes Protein oder Peptid kodiert.1 1. Expression vector according to one of Claims 8 to 10, characterized in that the polynucleotide encodes an antigenic protein or peptide.
12. Expressionsvektor nach Anspruch 1 1 , dadurch gekennzeichnet, daß das Polynucleotid ein Hepatitis-B-Surface-Antigen, VP1 aus Polyoma-Virus oder Protein A aus Staphylococcus kodiert.12. Expression vector according to claim 1 1, characterized in that the polynucleotide encodes a hepatitis B surface antigen, VP1 from polyoma virus or protein A from Staphylococcus.
13. Expressionsvektor nach einem der Ansprüche 8 bis 12, dadurch gekennzeichnet, daß der Vektor ein integrativer oder episomaler Vektor ist.13. Expression vector according to one of claims 8 to 12, characterized in that the vector is an integrative or episomal vector.
14. Expressionsvektor nach einem der Ansprüche 8 bis 12, dadurch gekennzeichnet, daß der Vektor ein in Hefe replizierbares Plasmid ist.14. Expression vector according to one of claims 8 to 12, characterized in that the vector is a plasmid replicable in yeast.
15. Wirtszelle transformiert mit einem Expressionsvektor oder einem Plasmid nach einem der vorhergehenden Ansprüche.15. Host cell transformed with an expression vector or a plasmid according to one of the preceding claims.
16. Wirtszelle nach Anspruch 14, dadurch gekennzeichnet, daß es eine Zelle der Art Kluyveromyces marxianus ist.16. Host cell according to claim 14, characterized in that it is a cell of the type Kluyveromyces marxianus.
17. E. coli pEPG1 -1 hinterlegt mit der Hinterlegungsnr. DSM 12919.17. E. coli pEPG1 -1 deposited with the deposit no. DSM 12919.
18. E. coli pUC19PG hinterlegt mit der Hinterlegungsnr. DSM 12920.18. E. coli pUC19PG deposited with the deposit no. DSM 12920.
19. E. coli pEPGSeq hinterlegt mit der Hinterlegungsnr. DSM 12921.19. E. coli pEPGSeq deposited with the deposit no. DSM 12921.
20. E. coli pEPG1 -2 hinterlegt mit der Hinterlegungsnummer DSM 12922.20. E. coli pEPG1 -2 deposited with the accession number DSM 12922.
21. Verfahren zur Herstellung eines rekombinanten Proteins, dadurch gekennzeichnet, daß man eine Hefezelle mit einem Plasmid, das die Expressionskassette nach einem der Ansprüche 2 oder 3 und ein Polynucleotid, das ein Fremdprotein kodiert, umfaßt, transfiziert oder transformiert, die Hefezelie unter Bedingungen, die zur Expression des Fremdproteins geeignet sind, züchtet und das Protein gewinnt. 21. A process for the production of a recombinant protein, characterized in that a yeast cell is transfected or transformed with a plasmid which comprises the expression cassette according to one of claims 2 or 3 and a polynucleotide which encodes a foreign protein, the yeast cell under conditions, which are suitable for the expression of the foreign protein, grow and the protein wins.
22. Verfahren zur Herstellung eines rekombinanten Proteins, dadurch gekennzeichnet, daß man eine Expressionskassette gemäß Anspruch 2 oder Anspruch 3 in eine Hefezelle bringt, wo die Expressionskassette in ein Chromosom eingebaut wird, die Zelle züchtet und anschließend das Protein gewinnt.22. A process for the production of a recombinant protein, characterized in that an expression cassette according to claim 2 or claim 3 is brought into a yeast cell, where the expression cassette is built into a chromosome, the cell is grown and then the protein is recovered.
23. Verwendung einer DNA-Sequenz gemäß SEQ ID Nr. 1 als Promotor zur Expression von Fremdprotein in Hefezellen. 23. Use of a DNA sequence according to SEQ ID No. 1 as a promoter for the expression of foreign protein in yeast cells.
EP00965925A 1999-09-10 2000-09-05 Regulatory sequences and expression cassettes for yeasts Withdrawn EP1214425A1 (en)

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EP2546340B1 (en) * 2010-02-09 2016-04-20 Yamaguchi University High expression promoter derived from kluyveromyces marxianus
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CN105063080A (en) * 2015-09-07 2015-11-18 复旦大学 Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain
CN105132452A (en) * 2015-09-07 2015-12-09 复旦大学 Recombinant vector for expressing histidine-tag-fused foreign gene in Kluyveromyces marxianus nutritional deficient strain
CN115976094B (en) * 2022-12-15 2024-02-20 浙江大学杭州国际科创中心 Genetically engineered bacterium for improving secretion of endogenous enzyme and construction method and application thereof

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US4943529A (en) * 1982-05-19 1990-07-24 Gist-Brocades Nv Kluyveromyces as a host strain
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AU5811194A (en) * 1992-12-11 1994-07-04 Quest International B.V. The use of the (kluyveromyces marxianus) inulinase gene promoter for protein production

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