EP1054990A2 - Expression vector for the production of dead proteins - Google Patents

Expression vector for the production of dead proteins

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Publication number
EP1054990A2
EP1054990A2 EP99911658A EP99911658A EP1054990A2 EP 1054990 A2 EP1054990 A2 EP 1054990A2 EP 99911658 A EP99911658 A EP 99911658A EP 99911658 A EP99911658 A EP 99911658A EP 1054990 A2 EP1054990 A2 EP 1054990A2
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EP
European Patent Office
Prior art keywords
protein
nucleic acid
vector according
insect
cells
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EP99911658A
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German (de)
French (fr)
Inventor
Karl-Christian Gallert
Stefan Müllner
Christoph Hüls
Britta BÖHNISCH
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Aventis Research and Technologies GmbH and Co KG
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Aventis Research and Technologies GmbH and Co KG
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Publication of EP1054990A2 publication Critical patent/EP1054990A2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to an insect cell vector for the production of proteins from the DEAD protein family.
  • RNA structure plays an essential function in cellular processes, e.g. in pre-mRNA splicing, in RNA transport or in protein translation, since the cellular RNA is present in the cell in different secondary and tertiary structures and, in addition, a large number of RNA-binding proteins ensure further structuring of the RNA.
  • modulation processes include Proteins from the family of the so-called DEAD box proteins are involved.
  • the members of this protein superfamily which as a characteristic contain a number of homologous protein sequences, so-called “protein boxes”, are named as motifs after the highly conserved tetrapeptide Asp-Glu-Ala-Asp, in the single-letter code D-E-A-D. About this protein superfamily also include several RNA or DNA helicases.
  • the characteristic protein sequences of the DEAD proteins are highly conserved in evolution.
  • a schematic representation of the proteins from the DEAD superfamily and their subfamilies according to FIG. 1 shows the similarity between the individual family members (see also Schmid, SR & Linder, P. (1992) Molecular Microbiology, 6, 283, No. 3; Fuller- Pace FV (1994) Trends in Cell Biology, 4, 271). It can be seen that the DEAD superfamily is divided into different subfamilies, which are named according to their sequence motif DEAH, DEXH or DEAH * subfamily. All family members have an ATP binding and RNA binding function as well as an ATP hydrolysis and RNA helicase function.
  • EP-A-0778347 describes a new ATP and nucleic acid binding protein with putative helicase and ATPase properties, which is assigned to the DEAH subfamily.
  • the RNA helicase also described together with the tolerance of certain cells to leflunomide and related compounds and is therefore suitable for the production of cell lines which are helpful in cancer, inflammation and apoptosis research and in the elucidation of mechanisms of action of medicinal substances.
  • Another possible application of this helicase is the identification of already known substances with regard to possible pharmaceutical properties such as an anticancerogenic or antiviral effect in a test or assay system. However, sufficient amounts of the protein are necessary for the desired uses of the RNA helicase.
  • RS domain a region between 50 and 200 amino acids in size, which has a high accumulation of argenin-serine repeats (one-letter code RS), is suspected to directly or indirectly make protein expression more difficult.
  • a direct effect can be caused, for example, by incorrect phosphorylation of the serine residues in this area.
  • overexpression of proteins with this domain can trigger toxic effects in the cell, since specific protein-protein interactions are mediated via this protein domain.
  • the native interaction via RS domains can be disturbed or inhibited.
  • RS proteins The family of RS proteins is a "subfamily" of proteins that is defined by the possession of the RS domain. These proteins are involved in a wide variety of processes of pre-mRNA splicing. RS domains can interact with proteins convey the RNA binding influence, modulate RNA-RNA annealing and act as subcellular localization signals. The relationship between the DEAD box and the RS proteins is that both are involved in the modulation of RNA structure and function and therefore many proteins can be assigned to the protein families.
  • the RS domain in human RNA helicase according to SEQ ID No. 7 lies in the range from approx. 131 to approx. 253 and in particular in the range from approx. 175 to approx. 216 based on the amino acid position.
  • the present invention therefore relates to an insect cell vector containing a nucleic acid coding for a protein from the DEAD protein superfamily.
  • nucleic acid is preferably understood to mean single- or double-stranded DNA or RNA, in particular double-stranded DNA.
  • the coding nucleic acid at the 3'-end of the coding region additionally contains a native 3'-non-coding region, which in preferred embodiments is at least approximately 50, preferably approximately 50 to approximately 450, in particular approximately 50 to is approximately 400 nucleotides long.
  • “Native” in the sense of the present invention refers to 3′-non-coding nucleic acid regions which come from the same organism, preferably from the same gene as the coding nucleic acid. If, for example, the nucleic acid codes for a human RNA helicase according to EP-A-0778347, the 3 ′ non-coding region according to the preferred embodiment likewise originates from human cells, in particular from the gene coding for the designated RNA helicase. The 3 'non-coding region according to SEQ ID No. 10th
  • CPSF Cleavage and Polyadenylation Specificity Factor
  • the CPSF protein consists of a complex of subunits with molecular weights of 160, 100, 73 and 30 kD.
  • CstF cleavage stimulation factor
  • This protein is a heterotrimer consisting of three subunits with 77, 64 and 50 kD.
  • CstF cleavage stimulation factor
  • the poly (A) polymerase is a polypeptide with a molecular mass of 83 kD.
  • the polymerase is both attached the poly (A) tail synthesis, as well as involved in its cleavage.
  • the extension of the poly (A) tail is strongly stimulated by the so-called "poly (A) binding protein II” (PABII).
  • PABII poly (A) binding protein II
  • Further information and references can be found in Wahl, E. (1995) Biochemica at Biophysica Acta, 1261, 183. Thus Wahl, E. (1995) describes e.g. in addition to the AAUAAA binding sequence, other consensus motifs such as a GU-rich region with the proposed consensus sequence YGUGUUYY and U-rich elements (see also Proudfoot, N. (1991) Cell, 64, 671-674).
  • the present invention therefore relates to 3 'non-coding regions which form a binding site for the CPSF protein, the CstF protein, the CF I protein, the CF II protein, the poly (A) Polymerase and / or the poly (A) binding protein II (PABII) contains, such as, for example, an AATAAA binding site designated in the form of its cDNA form, ATTAAA binding site, a GT-rich element, in particular a YGTGTTY ⁇ element, and / or a T-rich element.
  • a protein from the DEAD protein superfamily is understood to mean proteins which have conserved motifs, of which a conserved motif contains the amino acid sequence DEAD, DEAH or DEXH.
  • the proteins preferably contain sequence motifs which are responsible for a nucleic acid-binding activity, a helicase activity and / or an ATPase activity.
  • the proteins contain RNA helicase and ATPase activity.
  • FIG. 1 and FIG. 2 show examples of the conserved motifs for the DEAD protein superfamily and the DEAH, DEXH or DEAH * subfamilies.
  • DEAD protein superfamily thus includes, within the meaning of the present invention, all proteins which fall under a group according to FIG. 1 or 2. Examples of such proteins are in Fuller-Pace, FV. (1994), supra, and Schmid , SR and Linder, P. (1992), Supra, further preferred proteins are those which give cells tolerance to isoxazole derivatives, such as leflunomide, and activity-related compounds, such as brequinar, and human proteins are particularly preferred, in particular those from Table 1 and the RNA helicase from EP-A-0778347.
  • proteins with a molecular mass of approximately 100 to approximately 150 kD, in particular with a molecular mass of approximately 130 kD are preferably suitable and such with a so-called SR domain, ie an area of approximately 50 to 200 amino acids with an accumulation of arginine-tendon repeats .
  • a nucleic acid codi is particularly suitable for the purposes of the present invention for human RNA helicase p135 according to EP 0778347 with the amino acid sequence according to FIG. 3. Table 1
  • SNF2L1 SNF2L1 (SMBP2) P28370 *; L24544
  • DDX9 (RNA helicase A) L13848; Y10658
  • nucleic acid coding for a protein from the DEAH protein subfamily with a native 3 'non-coding region is the cDNA of human RNA helicase from EP-A-0778347 according to FIG. 5 of the present invention.
  • the 3 'non-coding region of the said RNA helicase according to SEQ ID No. 10 is generally suitable in the sense of the present invention as a native 3'-non-coding region of human proteins from the DEAD protein superfamily and in particular from the DEAH protein subfamily.
  • the vector according to the invention contains regulatory sequences which control the expression of the nucleic acid coding for a protein from the DEAD protein superfamily. All regulatory sequences known to the person skilled in the art are suitable for this.
  • regulatory sequences from insect viruses preferably baculoviruses, in particular the promoter of polyhedra wrestling or 10K
  • the native ATG start codon of the nucleic acid coding for a protein from the DEAD protein superfamily is replaced by a polyhedrin ATG translation initiation start site
  • the invention is thus a chimeric nucleic acid from insect virus sequences on the 5'- End and downstream subsequent heterologous sequences, the 3 'non-coding part preferably containing native sequences to the heterologous part.
  • This construct according to the invention enables a further advantageous increase in expression in insect cells.
  • the nucleic acid according to the invention contains a nucleic acid coding for an oligopeptide of at least about 4, preferably of about 6, histidines between the ATG translation initiation start site and the region coding for the protein from the DEAD protein superfamily.
  • a fusion protein is obtained from the selected protein from the DEAD protein superfamily and an N-terminally fused peptide which contains the histidines mentioned.
  • the protein can be obtained in a particularly simple and effective manner, for example using a chromatography column containing metal ions, e.g. Clean a chromatography column containing nickel, such as a chromatography column containing Ni-NTA resin.
  • NTA stands for the chelator “nitrilotriacetic acid” (Qiagen GmbH, Hilden).
  • a nucleic acid which codes for the glutathione-S-transferase Smith, D.B. & Johnson, K.S. (1988) Gene, 67, 31-40
  • the fusion proteins obtained in this way can also be purified in a simple manner using affinity chromatography and detected using a colorimetric test or an immunoassay.
  • a suitable system is, for example, the vector pGEX from Pharmacia, Freiburg as the starting vector.
  • Suitable proteases are, for example, thrombin or factor Xa.
  • the thrombin cleavage site contains, for example, the amino acid sequence Leu-Val-Pro-Arg-Gly-Ser (see, for example, FIG. 3B).
  • the factor Xa cleavage site contains, for example, the amino acid sequence Ile-Glu-Gly-Arg.
  • a preferred 5 'region of the nucleic acid according to the present invention is, for example, a nucleic acid according to FIG.
  • a nucleic acid suitable according to the invention is a nucleic acid containing the polyhedrin promoter, e.g. B. according to EP-B1-0 127 839, the nucleic acid p135-NT5C according to SEQ ID No. 12 containing the polyhedrin ATG translation initiation site and a sequence coding for 6 histidines and a nucleic acid according to SEQ ID No. 9 containing a nucleic acid coding for the RNA helicase p135 and its native 3'-non-coding region.
  • the 5 ′ region of the nucleic acid according to the invention contains a nucleic acid which is suitable for a signal sequence, for example an insulin signal sequence, for example according to SEQ ID No. 13 in the form of the construct p135-NT5S, coded.
  • a signal sequence for example an insulin signal sequence, for example according to SEQ ID No. 13 in the form of the construct p135-NT5S, coded.
  • This construct also has the advantage that the desired protein can be worked up and purified particularly easily, since it is secreted directly into the culture medium due to the signal sequence and the signal sequence is thereby split off instead of accumulating the desired protein intracellularly in the insect cells.
  • Other suitable signal sequences are the signal sequence of silk worm bombyxin (Congote, LF & Li, Q. (1994) Biochem.
  • Another object of the present invention is a method for producing recombinant insect viruses which code for a protein from the DEAD protein superfamily according to the present invention, in which a vector according to the invention is introduced into insect cells together with insect virus wild-type DNA, and the resulting recombinants Insect viruses to be isolated.
  • a suitable insect virus is, for example, the baculovirus, in particular the Autographa Californica virus.
  • suitable insect cells are Spodoptera Frugiperda, Trichoplusia ni, Rachiplusia ou or Galleria Mellonela.
  • the Autographa Californica strains E2, R9, S1 or S3 are particularly suitable, especially the Autographa Californica strain S3, Spodoptera Frugiperda strain 21 or Trichoplusia ni egg cells.
  • oval cells of the corresponding insects or their larvae are also suitable.
  • the recombinant insect virus according to the invention arises in the insect cells by homologous recombination of the vector according to the invention with the wild-type insect virus in question (see, for example, EP-B1-0127839 or U.S. Pat. 5,004,687).
  • the recombinant insect virus can then be used to produce the desired protein.
  • Another object of the present invention therefore also relates to a method for producing a protein from the DEAD protein superfamily, in which a vector or a recombinant insect virus according to the invention is introduced into insect cells or insect larvae, which cultivates the insect cells or larvae under suitable conditions and the expressed protein is isolated.
  • Insect cells are preferably infected with recombinant insect virus, the duration of infection preferably being about 40 to about 90, in particular about 70 hours.
  • the production of a recombinant insect virus or the production of a desired protein in insect cells is carried out by methods which are generally known to the person skilled in the art, such as are described, for example, in EP-B1-0127839 or US Pat. No. 5,004,687. It is suitable however, also commercially available baculovirus expression systems such as, for example, the Baculo Gold TM transfection kit from Pharmingen or the Bac-to-Bac TM baculovirus expression system from Gibco BRL.
  • An advantage of the insect cell expression vectors according to the invention or the methods according to the invention is that, surprisingly, larger amounts, generally about 300-400 mg per 10 9 cells, of proteins from the DEAD protein superfamily, in particular of proteins with a molecular mass of> about 100 kD and especially proteins with a so-called SR domain can be produced.
  • Another object of the present invention therefore relates to the use of an insect cell vector according to the invention for producing a protein from the DEAD protein superfamily.
  • the designated proteins are suitable, for example, for the production of corresponding test systems according to EP-A-0778347 or for the treatment of a disease as in EP-A-0778347 or in Ellis N.A. (1997), supra.
  • FIG. 1 shows schematically the conserved areas of the proteins from the DEAD protein superfamily and the DEAH and DEXH subfamilies, and, as an example, the conserved areas of the protein elF-4A.
  • the numbers between the areas indicate the distances in amino acids.
  • X stands for any amino acid.
  • FIG. 2 schematically describes the conserved areas and their known functions of the proteins for the DEAD, DEAH, DEXH and DEAH * families, according to Fuller-Pace, FV (1994), supra.
  • SEQ ID No. 7 shows the amino acid sequence of the human RNA helicase p135.
  • the RS domain is position 131 to 253.
  • SEQ ID No. 8 shows the nucleic acid sequence of the human RNA helicase p135.
  • SEQ ID No. 9 shows the nucleic acid sequence of the human RNA helicase p135 including its 3 'non-coding region.
  • SEQ ID No. 10 is p135-NT3 SEQ ID No. 11 is p135-Pi3 SEQ ID No. 12 is p135-NT5C SEQ ID No. 13 is p135-NT5S SEQ ID No. 14 is p135-NTPS SEQ ID No. 15 is p135-NTGEX.
  • FIGS. 3A and B schematically show the pAcHLT-A baculovirus transfer vector and coding sequences of the foreign component in the fusion protein (Invitrogen®). 3 B corresponds to SEQ ID No. 16 and SEQ ID No. 17
  • Figures 4 A and B schematically show the pFASTBACI baculovirus transfer vector and the cloning site (Gibco-BRL). 4 B corresponds to SEQ ID No. 18th
  • FIG. 5 shows schematically the production of the vector KL33.
  • p135-CDS means the coding p135-DNA sequence from the 2nd coding base triplet to the last coding base triplet.
  • p135-GS means the coding p135-DNA sequence from the 2nd coding base triplet to the last base of the 3'-untranslated region.
  • FIG. 6 shows an overview of the gene constructs used for the expression of the p135 protein in different host cells.
  • FIG. 7 shows an overview of deletion constructs of p135.
  • N1 5 ' -ATGAATTCGGGGACACCAGTGAGGATGCCTCG-3 ' (SEQ ID No. 3) and N2: 5 ' -CCGATAATGTCTGTCTTTCCGGATATT-3 ' (SEQ ID No. 4)
  • the PCR fragment together with the BspEI-Notl fragment of the cDNA of the p135 protein was used in a ligation reaction with the vector pAcHLT-A, which was linearized with the enzymes EcoRI and Notl.
  • the plasmid KL33 thus obtained was by DNA sequencing confirmed.
  • the 5'-untranslated region of the p135 cDNA is replaced by a so-called hexahistidine tag.
  • This sequence section encodes a sequence of 6 histidine residues which facilitate the detection and the purification of the fusion protein obtained.
  • a short section of the 3 'non-coding DNA is also inserted between the stop codon of the p135 cDNA and the terminator sequence specified by the vector pAcHLT-A.
  • the plasmid KL33 was used in a baculovirus co-transfection.
  • a plaque test was then carried out to identify recombinant baculoviruses.
  • isolated plaques were then incubated with 1x10 6 SF21 cells (Invitrogen ®).
  • the virus DNA was then isolated and used as a template for a PCR.
  • the oligodeoxynucleotides N1 and N2 were used in this test PCR. Only in the case of recombinant baculoviruses, but not in the case of wild-type baculoviruses, a band of approx. 310 bp was found when analyzing the PCR batches in the agarose gel. Clone KL33 was sequenced for further confirmation.
  • Well-grown SF21 cultures (approx. 2 ⁇ 10 7 cells) were infected with 200 ⁇ l of recombinant viruses in 75 cm 2 tissue culture areas and incubated for 7 days at 27 ° C. in order to obtain sufficient BV33 stock for the subsequent protein expressions in Trichoplusia ni egg cells (“ High-Five-Cells ", Invitrogen ® ).
  • 3 ml of this stock solution in 100 ml SF21 culture (* 2 ⁇ 10 8 cells) in 250 ml spinner (Technomara ®) were incubated for 7 days at 27 ° C. for further propagation.
  • the virus titer of the BV33 stock solution was determined using the virus titer assay.
  • the p135 DNA was cloned into the vector pFASTBACI (Gibco-BRL) (see FIGS. 4A and B).
  • the insulin signal sequence (p135-NT5S, SEQ ID No. 12) obtained from the hybridization of synthetic oligodeoxynucleotides and subsequent fill-in synthesis was first cloned in via the BamHi and EcoRI interface of this vector.
  • the vector FB1 thus obtained was then linearized by restriction with EcoRI and NotI.
  • the vector thus linearized was incubated together with the sequence p135-GS from the vector KL33 in the presence of T4 ligase.
  • the protein p135 was obtained from the supernatants of infected HF cells (grown in a medium as described in Example 1, but without FCS) by concentrating the supernatant and subsequent chromatographic purification with appropriate homogeneity. The secretion into the culture medium made the processing and cleaning considerably easier.
  • E.coii TOP10 cells (Invitrogen ® ) were transformed with the two clones Ec33-N and Ec33-M.
  • the transformed cells obtained were, after confirmation of the successful transformation, minilysed to an OD800 of 0.7 and then induced with IPTG. After 1, 2, 3 and 4 hours, aliquots were analyzed for expression in SDS-PAGE. No expression products could be detected.
  • deletion constructs D1 and D2 were expressed as described above (see FIG. 7). Expression products could be detected with construct D2, which represents an internal fragment.
  • the P. pastoris cells from the KM71 strain were transformed according to the manufacturer's instructions (Invitrogen ® ). Likewise, for the analysis of the Proceed protein expression according to the manufacturer (Invitrogen ® , No. K1740-01). No expression products could be detected.
  • deletion clones D1 and D2 were successfully expressed as described above.

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Abstract

Die Erfindung bezieht sich auf einen Insektenzell-Vektor zur Herstellung von Proteinen aus der DEAD-Proteinfamilie.

Description

Expressionsvektor zur Herstellung von DEAD-ProteinenExpression vector for the production of DEAD proteins
Die vorliegende Erfindung betrifft einen Insekteπzell-Vektor zur Herstellung von Proteinen aus der DEAD-Proteinfamilie.The present invention relates to an insect cell vector for the production of proteins from the DEAD protein family.
Die Modulation der RNA-Struktur spielt eine essentielle Funktion in zellulären Vorgängen, wie z.B. beim prä-mRNA-Spleißen, beim RNA-Transport oder bei der Proteintranslation, da die zelluläre RNA in der Zelle in unterschiedlichen Sekundär- und Tertiärstrukturen vorliegt und daneben eine Vielzahl von RNA-bindender Proteine für eine weitere Strukturierung der RNA sorgt. An diesen Modulationsvorgängen sind u.a. Proteine aus der Familie der sogenannten DEAD- Box-Proteine beteiligt. Die Mitglieder dieser Proteinsuperfamilie, die als Charakte stikum eine Reihe homologer Proteinsequenzen, sogenannte „Proteinboxen" enthalten, sind nach dem hochkonservierten Tetrapeptid Asp-Glu- Ala-Asp, im Einbuchstabencode D- E- A- D, als Motiv benannt. Zu dieser Proteinsuperfamilie gehören auch mehrere RNA- bzw. DNA-Helicasen.The modulation of the RNA structure plays an essential function in cellular processes, e.g. in pre-mRNA splicing, in RNA transport or in protein translation, since the cellular RNA is present in the cell in different secondary and tertiary structures and, in addition, a large number of RNA-binding proteins ensure further structuring of the RNA. These modulation processes include Proteins from the family of the so-called DEAD box proteins are involved. The members of this protein superfamily, which as a characteristic contain a number of homologous protein sequences, so-called “protein boxes”, are named as motifs after the highly conserved tetrapeptide Asp-Glu-Ala-Asp, in the single-letter code D-E-A-D. About this protein superfamily also include several RNA or DNA helicases.
Die charakteristischen Proteinsequenzen der DEAD-Proteine sind in der Evolution hochkonserviert. Eine schematische Darstellung der Proteine aus der DEAD- Superfamilie und ihrer Unterfamilien gemäß Figur 1 zeigt die Ähnlichkeit zwischen den einzelnen Familienmitgliedern (siehe auch Schmid, S.R. & Linder, P. (1992) Molecular Microbiology, 6, 283, Nr. 3; Fuller-Pace F.V (1994) Trends in Cell Biology, 4, 271 ). Man erkennt, daß die DEAD-Superfamilie sich in verschiedene Unterfamilien gliedert, die nach ihrem Sequenzmotiv DEAH-, DEXH- oder DEAH*- Unterfamilie genannt werden. Alle Familienmitglieder besitzen eine ATP-Binde- und RNA-Bindefunktion sowie eine ATP-Hydrolyse- und RNA-Helicasefunktion.The characteristic protein sequences of the DEAD proteins are highly conserved in evolution. A schematic representation of the proteins from the DEAD superfamily and their subfamilies according to FIG. 1 shows the similarity between the individual family members (see also Schmid, SR & Linder, P. (1992) Molecular Microbiology, 6, 283, No. 3; Fuller- Pace FV (1994) Trends in Cell Biology, 4, 271). It can be seen that the DEAD superfamily is divided into different subfamilies, which are named according to their sequence motif DEAH, DEXH or DEAH * subfamily. All family members have an ATP binding and RNA binding function as well as an ATP hydrolysis and RNA helicase function.
In EP-A-0778347 wird nun ein neues ATP- und Nukleinsäure-bindendes Protein mit putativen Helicase- und ATPase-Eigenschaften beschrieben, welches der DEAH- Unterfamilie zugeordnet wird. Neben den genannten Eigenschaften hängt die beschriebene RNA-Helikase auch mit der Toleranz von bestimmten Zellen gegenüber Leflunomid und verwandten Verbindungen zusammen und eignet sich somit zur Herstellung von Zellinien, welche bei der Krebs-, Entzündungs- und Apoptose-Forschung sowie bei der Aufklärung von Wirkmechanismen von Arzneistoffen hilfreich sind. Eine weitere Anwendungsmöglichkeit dieser Helikase ist die Identifizierung bereits bekannter Substanzen bezüglich eventueller pharmazeutischer Eigenschaften wie z.B. einer anticancerogenen oder antiviralen Wirkung in einem Test- bzw. Assay-System. Für die gewünschten Verwendungsarten der RNA-Helicase sind jedoch ausreichende Mengen des Proteins nötig.EP-A-0778347 describes a new ATP and nucleic acid binding protein with putative helicase and ATPase properties, which is assigned to the DEAH subfamily. In addition to the properties mentioned, the RNA helicase also described together with the tolerance of certain cells to leflunomide and related compounds and is therefore suitable for the production of cell lines which are helpful in cancer, inflammation and apoptosis research and in the elucidation of mechanisms of action of medicinal substances. Another possible application of this helicase is the identification of already known substances with regard to possible pharmaceutical properties such as an anticancerogenic or antiviral effect in a test or assay system. However, sufficient amounts of the protein are necessary for the desired uses of the RNA helicase.
Interessanterweise ist es bis heute nicht gelungen, Proteine aus der DEAD- Proteinsuperfamilie in ausreichenden Mengen funktionell homolog oder heterolog zu exprimieren. Neben der Größe der Proteine, viele Vertreter dieser Familie besitzen eine Molekularmasse von 100 kD und darüber, scheinen bestimmte Strukturmotive die Expression in Fremdorganismen zu inhibieren. Insbesondere von der sogenannten RS-Domäne, einem zwischen 50 und 200 Aminosäuren großen Bereich, der eine starke Häufung von Argenin-Serin-Wiederholungeπ (Einbuchstabencode RS) aufweist, wird vermutet, daß sie die Proteinexpression direkt oder indirekt erschwert. Ein direkter Effekt kann beispielsweise durch falsche Phosphorylierung der Serin-Reste in diesem Bereich hervorgerufen werden. Indirekt kann eine Überexpressioπ von Proteinen mit dieser Domäne toxische Effekte in der Zelle auslösen, da über diese Proteindomäne spezifische Protein-Protein- Wechselwirkungen vermittelt werden. Im Falle der heterologen Proteinüberexpression kann so die native Interaktion über RS-Domänen gestört oder inhibiert sein.Interestingly enough, it has so far not been possible to express proteins from the DEAD protein superfamily in sufficient quantities in a functionally homologous or heterologous manner. In addition to the size of the proteins, many members of this family have a molecular mass of 100 kD and above, certain structural motifs seem to inhibit expression in foreign organisms. In particular, the so-called RS domain, a region between 50 and 200 amino acids in size, which has a high accumulation of argenin-serine repeats (one-letter code RS), is suspected to directly or indirectly make protein expression more difficult. A direct effect can be caused, for example, by incorrect phosphorylation of the serine residues in this area. Indirectly, overexpression of proteins with this domain can trigger toxic effects in the cell, since specific protein-protein interactions are mediated via this protein domain. In the case of heterologous protein overexpression, the native interaction via RS domains can be disturbed or inhibited.
Die Familie der RS-Proteine stellt eine „Unterfamilie" von Proteinen dar, die sich durch den Besitz der RS-Domäne definiert. Diese Proteine sind in den unterschiedlichsten Prozessen des pre-mRNA-Splicings beteiligt. RS-Domäne können Protein-Protein-Wechselwirkungen vermitteln, die RNA-Bindung beeinflussen, RNA-RNA-Annealiπg modulieren sowie als subzelluläre Lokalisationssignale fungieren. Der Zusammenhang zwischen den DEAD-Box- und den RS-Proteinen besteht darin, daß beide an der Modulation von RNA-Struktur- und Funktion beteiligt sind und daher viele Proteine bei den Proteinfamilien zuzuordnen sind.The family of RS proteins is a "subfamily" of proteins that is defined by the possession of the RS domain. These proteins are involved in a wide variety of processes of pre-mRNA splicing. RS domains can interact with proteins convey the RNA binding influence, modulate RNA-RNA annealing and act as subcellular localization signals. The relationship between the DEAD box and the RS proteins is that both are involved in the modulation of RNA structure and function and therefore many proteins can be assigned to the protein families.
Die RS-Domäne bei der humanen RNA-Helikase gemäß SEQ ID No. 7 liegt im Bereich von ca. 131 bis ca. 253 und insbesondere im Bereich von ca. 175 bis ca. 216 bezogen auf die Aminosäureposition.The RS domain in human RNA helicase according to SEQ ID No. 7 lies in the range from approx. 131 to approx. 253 and in particular in the range from approx. 175 to approx. 216 based on the amino acid position.
Aufgabe der vorliegenden Erfindung war es daher, ein Verfahren bereitzustellen, das die gentechnische Herstellung von Proteinen aus der DEAD-Proteinsuperfamilie in großen Mengen ermöglicht.It was therefore an object of the present invention to provide a process which enables the genetic engineering of proteins from the DEAD protein superfamily in large quantities.
Es wurde nun überraschenderweise gefunden, daß im Gegensatz zu der Expression in E. coli oder Hefe, die Expression in Insektenzellen auf vorteilhafte Weise gelingt.It has now surprisingly been found that, in contrast to expression in E. coli or yeast, expression in insect cells is advantageously achieved.
Ein Gegenstand der vorliegenden Erfindung ist daher ein Insektenzell-Vektor enthaltend eine Nukleinsäure kodierend für ein Protein aus der DEAD- Proteinsuperfamilie. Unter dem Begriff „Nukleinsäure" versteht man gemäß der vorliegenden Erfindung vorzugsweise einzel- oder doppelsträngige DNA oder RNA, insbesondere doppelsträngige DNA.The present invention therefore relates to an insect cell vector containing a nucleic acid coding for a protein from the DEAD protein superfamily. According to the present invention, the term “nucleic acid” is preferably understood to mean single- or double-stranded DNA or RNA, in particular double-stranded DNA.
In einer bevorzugten Ausführungsform enthält die kodierende Nukleinsäure am 3'- Ende des kodierenden Bereiches zusätzlich einen nativen 3'-nicht-kodierenden Bereich, welcher in bevorzugten Ausführungsformen mindestens ca. 50, vorzugsweise ca. 50 bis ca. 450, insbesondere ca. 50 bis ca. 400 Nukleotide lang ist.In a preferred embodiment, the coding nucleic acid at the 3'-end of the coding region additionally contains a native 3'-non-coding region, which in preferred embodiments is at least approximately 50, preferably approximately 50 to approximately 450, in particular approximately 50 to is approximately 400 nucleotides long.
„Nativ" im Sinne der vorliegenden Erfindung bezeichnet man 3'-nicht-kodiereπde Nukleinsäurebereiche, die aus dem selben Organismus, vorzugsweise aus dem selben Gen wie die kodierende Nukleinsäure stammt. Kodiert beispielsweise die Nukleinsäure für eine humane RNA-Helicase gemäß EP-A-0778347, so stammt der 3'-nicht-kodierende Bereich gemäß der bevorzugten Ausführungsform ebenso aus humanen Zellen, insbesondere aus dem Gen kodierend für die bezeichnete RNA- Helicase. Bevorzugt ist der 3'-nicht-kodierende Bereich gemäß SEQ ID No. 10.“Native” in the sense of the present invention refers to 3′-non-coding nucleic acid regions which come from the same organism, preferably from the same gene as the coding nucleic acid. If, for example, the nucleic acid codes for a human RNA helicase according to EP-A-0778347, the 3 ′ non-coding region according to the preferred embodiment likewise originates from human cells, in particular from the gene coding for the designated RNA helicase. The 3 'non-coding region according to SEQ ID No. 10th
Es ist bekannt, daß der 3'-nicht-kodierende Bereich von Genen verschiedene regulatorische Proteine bzw. Faktoren binden kann. So bindet beispielsweise der sogenannte „Cleavage and Polyadenylation Specificity Factor" (CPSF) an die nicht- kodierende RNA-Sequenz AAUAAA. Das CPSF-Protein besteht aus einem Komplex von Untereinheiten mit Molekulargewichten von 160, 100, 73 und 30 kD. Ein weiteres RNA-Bindeprotein ist der sogenannte „Cleavage Stimulation Factor" (CstF). Dieses Protein ist ein Heterotrimer aus drei Untereinheiten mit 77, 64 und 50 kD. Daneben gibt es weitere RNA-Bindeproteine wie die sogenannten „Cleavage Factors" CF I und CF II sowie eine Poly(A)-Polymerase. Die Poly(A)-Polymerase ist ein Polypeptid mit einer Molekularmasse von 83 kD. Die Polymerase ist sowohl an der Poly(A)-Schwanzsynthese, wie auch an deren Spaltung beteiligt. Die Verlängerung des Poly(A)-Schwaπzes wird stark durch das sogenannte „Poly(A)Bindingprotein II" (PABII) stimuliert. Weitere Informationen und Literaturhinweise finden sich in Wähle, E. (1995) Biochemica at Biophysica Acta, 1261 , 183. So beschreibt Wähle, E. (1995) z.B. neben der AAUAAA-Bindesequenz auch weitere Konsensusmotive wie eine GU-reiche Region mit der vorgeschlagenen Konsensussequenz YGUGUUYY sowie U-reiche Elemente (siehe auch Proudfoot, N. (1991 ) Cell, 64, 671 -674).It is known that the 3 'non-coding region of genes can bind various regulatory proteins or factors. For example, the so-called "Cleavage and Polyadenylation Specificity Factor" (CPSF) binds to the non-coding RNA sequence AAUAAA. The CPSF protein consists of a complex of subunits with molecular weights of 160, 100, 73 and 30 kD. Another RNA Binding protein is the so-called "cleavage stimulation factor" (CstF). This protein is a heterotrimer consisting of three subunits with 77, 64 and 50 kD. There are also other RNA binding proteins such as the so-called "cleavage factors" CF I and CF II and a poly (A) polymerase. The poly (A) polymerase is a polypeptide with a molecular mass of 83 kD. The polymerase is both attached the poly (A) tail synthesis, as well as involved in its cleavage. The extension of the poly (A) tail is strongly stimulated by the so-called "poly (A) binding protein II" (PABII). Further information and references can be found in Wahl, E. (1995) Biochemica at Biophysica Acta, 1261, 183. Thus Wahl, E. (1995) describes e.g. in addition to the AAUAAA binding sequence, other consensus motifs such as a GU-rich region with the proposed consensus sequence YGUGUUYY and U-rich elements (see also Proudfoot, N. (1991) Cell, 64, 671-674).
In einer bevorzugten Ausführungsform bezieht sich die vorliegende Erfindung daher auf 3'-nicht-kodierende Bereiche, die eine Bindestelle für das CPSF-Protein, das CstF-Protein, das CF I-Protein, das CF Il-Protein, die Poly(A)-Polymerase und/oder das Poly(A)-Bindeprotein II (PABII) enthält, wie z.B. eine in Form ihrer cDNA-Form bezeichnete AATAAA-Bindestelle, ATTAAA-Bindestelle, ein GT-reiches Element, insbesondere ein YGTGTTYΥ-Element, und/oder ein T-reiches Element. Unter einem Protein aus der DEAD-Proteinsuperfamilie versteht man gemäß der vorliegenden Erfindung Proteine, die konservierte Motive besitzen, worunter ein konserviertes Motiv die Aminosäurereihenfolge DEAD, DEAH oder DEXH enthält. Vorzugsweise enthalten die Proteine Sequenzmotive, die für eine Nukleinsäure- bindende Aktivität, eine Helicase-Aktivität und/oder eine ATPase-Aktivität verantwortlich sind. Insbesondere enthalten die Proteine eine RNA-Helicase- und ATPase-Aktivität. Beispiele der konservierten Motive zu der DEAD- Proteinsuperfamilie und der DEAH-, DEXH- bzw. DEAH*-Unterfamiiien zeigt Figur 1 und Figur 2.In a preferred embodiment, the present invention therefore relates to 3 'non-coding regions which form a binding site for the CPSF protein, the CstF protein, the CF I protein, the CF II protein, the poly (A) Polymerase and / or the poly (A) binding protein II (PABII) contains, such as, for example, an AATAAA binding site designated in the form of its cDNA form, ATTAAA binding site, a GT-rich element, in particular a YGTGTTYΥ element, and / or a T-rich element. According to the present invention, a protein from the DEAD protein superfamily is understood to mean proteins which have conserved motifs, of which a conserved motif contains the amino acid sequence DEAD, DEAH or DEXH. The proteins preferably contain sequence motifs which are responsible for a nucleic acid-binding activity, a helicase activity and / or an ATPase activity. In particular, the proteins contain RNA helicase and ATPase activity. FIG. 1 and FIG. 2 show examples of the conserved motifs for the DEAD protein superfamily and the DEAH, DEXH or DEAH * subfamilies.
Der Begriff „DEAD-Proteinsuperfamilie" schließt somit im Sinne der vorliegenden Erfindung alle Proteine, die unter eine Gruppe gemäß Fig. 1 oder 2 fallen, ein. Beispiele derartiger Proteine sind in Fuller-Pace, FV. (1994), supra, und Schmid, S.R. und Linder, P. (1992), supra, beschrieben. Weitere bevorzugte Proteine sind solche, welche Zellen Toleranz gegenüber Isoxazol-Derivate, wie z.B. Leflunomid, und wirkungsverwandte Verbindungen, wie z.B. Brequinar, verleihen. Besonders bevorzugt sind humane Proteine, insbesondere solche aus der Tabelle 1 und die RNA-Helicase aus EP-A-0778347. Vorzugsweise eignen sich im Sinne der vorliegenden Erfindung Proteine mit einer Molekularmasse von ca. 100 bis ca. 150 kD, insbesondere mit einer Molekularmasse von ca. 130 kD und solche mit einer sogenannten SR-Domäne, d.h. einem ca. 50 bis 200 Aminosäuren großen Bereich mit einer Häufung von Arginin-Sehn-Wiederholungen. Insbesondere eignet sich im Sinne der vorliegenden Erfindung eine Nukleinsäure kodierend für die humane RNA-Helicase p135 gemäß EP 0778347 mit der Aminosäuresequenz gemäß Figur 3. Tabelle 1The term “DEAD protein superfamily” thus includes, within the meaning of the present invention, all proteins which fall under a group according to FIG. 1 or 2. Examples of such proteins are in Fuller-Pace, FV. (1994), supra, and Schmid , SR and Linder, P. (1992), Supra, further preferred proteins are those which give cells tolerance to isoxazole derivatives, such as leflunomide, and activity-related compounds, such as brequinar, and human proteins are particularly preferred, in particular those from Table 1 and the RNA helicase from EP-A-0778347. For the purposes of the present invention, proteins with a molecular mass of approximately 100 to approximately 150 kD, in particular with a molecular mass of approximately 130 kD, are preferably suitable and such with a so-called SR domain, ie an area of approximately 50 to 200 amino acids with an accumulation of arginine-tendon repeats .. A nucleic acid codi is particularly suitable for the purposes of the present invention for human RNA helicase p135 according to EP 0778347 with the amino acid sequence according to FIG. 3. Table 1
Humane Helicase-GeneHuman helicase genes
Gen (oder Protein) GenBank-Zuqanqsnummer DEXH-Box DNA HelicaseGene (or protein) gene bank number DEXH-Box DNA helicase
XPB; ERCC3 M31899XPB; ERCC3 M31899
XPD; ERCC2 X52221 ; L47234XPD; ERCC2 X52221; L47234
DDW11 (CHLR1 ) U33833DDW11 (CHLR1) U33833
DDX12 (CHLR2) U33834DDX12 (CHLR2) U33834
RECQL L36140; D37984RECQL L36140; D37984
BLM U39817BLM U39817
WRN L76937WRN L76937
CSB; ERCC6 L04791COD; ERCC6 L04791
ATRX U09820; U72936-U72938ATRX U09820; U72936-U72938
HRAD54 X97795HRAD54 X97795
SNF2L1 (SMBP2) P28370*; L24544SNF2L1 (SMBP2) P28370 *; L24544
SNF2L2 (HBRM) X72889SNF2L2 (HBRM) X72889
SNF2L3 (HIP 116; HTLF) Z46606SNF2L3 (HIP 116; HTLF) Z46606
SNF2L4 (BRG-1) U29175SNF2L4 (BRG-1) U29175
DEAD-Box RNA HelicaseDEAD box RNA helicase
DDX5 (p68) X15729; X52104DDX5 (p68) X15729; X52104
DDX6 (RCK; p54) Z11685; D17532DDX6 (RCK; p54) Z11685; D17532
(P72) U59321(P72) U59321
(BAT1) Z37166(BAT1) Z37166
(MRDB) X98743(MRDB) X98743
DDX10 U28042DDX10 U28042
(Gu; RNA Helicase II) U41387(Gu; RNA Helicase II) U41387
DDX7 (NP52) D26528DDX7 (NP52) D26528
EIF4A (elF4A-1) D30655EIF4A (elF4A-1) D30655
(elF4A-like) P38919* DDX1 (cl. 1042) X70649(elF4A-like) P38919 * DDX1 (cl. 1042) X70649
DEXH-Box RNA HelicaseDEXH box RNA helicase
DDX9 (RNA Helicase A) L13848; Y10658DDX9 (RNA helicase A) L13848; Y10658
DDX8 (HRH1) D50487DDX8 (HRH1) D50487
SKIV2L (SKI2W; 170 A) Z48796SKIV2L (SKI2W; 170 A) Z48796
KIAA0134 D50924KIAA0134 D50924
(Mi-2) X86691(Mi-2) X86691
*Swiss-Prot-Zugangsnummer* Swiss Prot access number
(aus Ellis, N.A. (1997), Current Opinion in Genetics & Development, 7, 354)(from Ellis, N.A. (1997), Current Opinion in Genetics & Development, 7, 354)
Ein weiteres bevorzugtes Beispiel einer Nukleinsäure kodierend für ein Protein aus der DEAH-Proteinunterfamilie mit einem nativen 3'-nicht-kodierenden Bereich ist die cDNA der humanen RNA-Helicase aus EP-A-0778347 gemäß Figur 5 der vorliegenden Erfindung. Der 3'-nicht-kodierende Bereich der genannten RNA- Helicase gemäß SEQ ID No. 10 eignet sich allgemein im Sinne der vorliegenden Erfindung als nativer 3'-nicht-kodierender Bereich humaner Proteine aus der DEAD- Proteinsuperfamilie und insbesondere aus der DEAH-Proteinunterfamilie.Another preferred example of a nucleic acid coding for a protein from the DEAH protein subfamily with a native 3 'non-coding region is the cDNA of human RNA helicase from EP-A-0778347 according to FIG. 5 of the present invention. The 3 'non-coding region of the said RNA helicase according to SEQ ID No. 10 is generally suitable in the sense of the present invention as a native 3'-non-coding region of human proteins from the DEAD protein superfamily and in particular from the DEAH protein subfamily.
In einer bevorzugten Ausführungsform enthält der erfindungsgemäße Vektor regulatorische Sequenzen, die die Expression der Nukleinsäure kodierend für ein Protein aus der DEAD-Proteinsuperfamilie steuern. Hierzu eignen sich alle dem Fachmann bekannten regulatorischen Sequenzen. Besonders geeignet sind ein Promotor eines „Long terminal repeat" (LTR) insbesondere eines retroviralen LTRs oder eines LTRs eines transposablen Elementes gemäß beispielsweise U.S. Pat. 5,004,687. Besonders bevorzugt sind regulatorische Sequenzen aus Insektenviren, vorzugsweise Baculoviren, insbesondere der Promotor des Polyhedringens oder des 10K Proteins (siehe z.B. EP-B1 -0127839). In einer weiteren bevorzugten Ausführungsform ist das native ATG-Startkodon der Nukleinsäure kodierend für ein Protein aus DEAD-Proteinsuperfamilie ersetzt durch eine Polyhedrin-ATG- Translations-Initiations-Startstelle. Die Nukleinsäure gemäß der vorliegenden Erfindung ist somit eine chimäre Nukleinsäure aus Insektenvirussequenzen am 5'- Ende und stromabwärts anschließende heterologe Sequenzen, wobei der 3'-nicht- kodierende Teil vorzugsweise zum heterologen Teil native Sequenzen enthält. Dieses erfindungsgemäße Konstrukt ermöglicht eine weitere vorteilhafte Expressionssteigerung in Insektenzellen.In a preferred embodiment, the vector according to the invention contains regulatory sequences which control the expression of the nucleic acid coding for a protein from the DEAD protein superfamily. All regulatory sequences known to the person skilled in the art are suitable for this. A promoter of a "long terminal repeat" (LTR), in particular a retroviral LTR or an LTR of a transposable element according to, for example, US Pat. No. 5,004,687, is particularly suitable. Particularly preferred are regulatory sequences from insect viruses, preferably baculoviruses, in particular the promoter of polyhedra wrestling or 10K In a further preferred embodiment, the native ATG start codon of the nucleic acid coding for a protein from the DEAD protein superfamily is replaced by a polyhedrin ATG translation initiation start site The invention is thus a chimeric nucleic acid from insect virus sequences on the 5'- End and downstream subsequent heterologous sequences, the 3 'non-coding part preferably containing native sequences to the heterologous part. This construct according to the invention enables a further advantageous increase in expression in insect cells.
In einer anderen bevorzugten Ausführungsform enthält die erfindungsgemäße Nukleinsäure zwischen der ATG-Translations-Initiations-Startstelle und dem für das Protein aus der DEAD-Proteinsuperfamilie kodierenden Bereich eine Nukleinsäure kodierend für ein Oligopeptid aus mindestens ca. 4, vorzugsweise aus ca. 6 Histidinen. Nach Expression der bezeichneten Nukleinsäure erhält man ein Fusionsprotein aus dem gewählten Protein aus der DEAD-Proteinsuperfamilie und einem N-terminal fusionierten Peptid, das die genannten Histidine enthält. Hierdurch läßt sich das Protein auf besonders einfache und effektive Weise beispielsweise über eine metallionhaltige Chromatographiesäule, wie z.B. eine πickelhaltige Chromatographiesäule, wie Ni-NTA-Harz-haltige Chromatographiesäule, reinigen. „NTA" steht für den Chelator „nitrilotriacetic acid" (Qiagen GmbH, Hilden). Anstelle oder zusätzlich zur Nukleinsäure kodierend für die genannten Histidine kann auch eine Nukleinsäure verwendet werden, die für die Glutathion-S-transferase (Smith, D.B. & Johnson, K.S. (1988) Gene, 67, 31-40) kodiert. Die so erhaltenen Fusionsproteine können ebenfalls auf einfache Weise über Affinitätschromatographie gereinigt und über einen kolorimetrischen Test oder über einen Immunoassay nachgewiesen werden. Ein geeignetes System ist beispielsweise der Vektor pGEX der Fa. Pharmacia, Freiburg als Ausgangsvektor.In another preferred embodiment, the nucleic acid according to the invention contains a nucleic acid coding for an oligopeptide of at least about 4, preferably of about 6, histidines between the ATG translation initiation start site and the region coding for the protein from the DEAD protein superfamily. After expression of the designated nucleic acid, a fusion protein is obtained from the selected protein from the DEAD protein superfamily and an N-terminally fused peptide which contains the histidines mentioned. As a result, the protein can be obtained in a particularly simple and effective manner, for example using a chromatography column containing metal ions, e.g. Clean a chromatography column containing nickel, such as a chromatography column containing Ni-NTA resin. "NTA" stands for the chelator "nitrilotriacetic acid" (Qiagen GmbH, Hilden). Instead of or in addition to the nucleic acid coding for the histidines mentioned, a nucleic acid which codes for the glutathione-S-transferase (Smith, D.B. & Johnson, K.S. (1988) Gene, 67, 31-40) can also be used. The fusion proteins obtained in this way can also be purified in a simple manner using affinity chromatography and detected using a colorimetric test or an immunoassay. A suitable system is, for example, the vector pGEX from Pharmacia, Freiburg as the starting vector.
Zur Abspaltung des Fremdproteinanteils aus dem genannten Fusionsproteiπ ist es vorteilhaft, wenn die Nukleinsäure für eine Protease-Schnittstelle kodiert. Geeignete Proteasen sind beispielsweise Thrombin, oder Faktor Xa. Die Thrombinspaltstelle enthält beispielsweise die Aminosäuresequenz Leu-Val-Pro-Arg-Gly-Ser (siehe z.B. Figur 3B). Die Faktor Xa-Spaltstelle enthält beispielsweise die Aminosäuresequenz Ile-Glu-Gly-Arg. Ein bevorzugter 5'-Bereich der Nukleinsäure gemäß der vorliegenden Erfindung ist beispielsweise eine Nukleinsäure gemäß Figur 3B, welche beim Startkodon ATG beginnt und nach der Thrombinspaltstelle an einer der bezeichneten Restriktioπsenzymschnittstellen endet. An die ausgewählte Restriktionsenzymschnittstelle kann dann nach allgemein bekannten Verfahren die betreffende Nukleinsäure ligiert werden. Eine erfindungsgemäß geeignete Nukleinsäure ist eine Nukleinsäure enthaltend den Polyhedrinpromotor, z. B. gemäß EP-B1-0 127 839, die Nukleinsäure p135-NT5C gemäß SEQ ID No. 12 enthaltend die Polyhedrin-ATG-Translations-Initiations-Startstelle und eine Sequenz kodierend für 6 Histidine und eine Nukleinsäure gemäß SEQ ID No. 9 enthaltend eine Nukleinsäure kodierend für die RNA-Helicase p135 und deren nativen 3'-nicht- kodierenden Bereich.To split off the foreign protein portion from the fusion protein mentioned, it is advantageous if the nucleic acid codes for a protease interface. Suitable proteases are, for example, thrombin or factor Xa. The thrombin cleavage site contains, for example, the amino acid sequence Leu-Val-Pro-Arg-Gly-Ser (see, for example, FIG. 3B). The factor Xa cleavage site contains, for example, the amino acid sequence Ile-Glu-Gly-Arg. A preferred 5 'region of the nucleic acid according to the present invention is, for example, a nucleic acid according to FIG. 3B, which begins at the start codon ATG and ends after the thrombin cleavage site at one of the designated restriction enzyme interfaces. The nucleic acid in question can then be ligated to the selected restriction enzyme interface by generally known methods. A nucleic acid suitable according to the invention is a nucleic acid containing the polyhedrin promoter, e.g. B. according to EP-B1-0 127 839, the nucleic acid p135-NT5C according to SEQ ID No. 12 containing the polyhedrin ATG translation initiation site and a sequence coding for 6 histidines and a nucleic acid according to SEQ ID No. 9 containing a nucleic acid coding for the RNA helicase p135 and its native 3'-non-coding region.
In einer weiteren bevorzugten Ausführungsform enthält der 5'-Bereich der erfindungsgemäßen Nukleinsäure eine Nukleinsäure, die für eine Signalsequenz, beispielsweise eine Insulinsigπalsequenz z.B. gemäß SEQ ID No. 13 in Form des Konstruktes p135-NT5S, kodiert. Auch dieses Konstrukt hat den Vorteil, daß das gewünschte Protein besonders leicht aufgearbeitet und gereinigt werden kann, da es aufgrund der Signaisequenz direkt in das Kulturmedium sekretiert und dabei die Signalsequeπz abgespalten wird, anstatt das gewünschte Protein intrazellulär in den Insektenzellen anzuhäufen. Weitere geeignete Signalsequenzen sind die Signalsequenz von Bombyxin vom Seidenwurm (Congote, L.F. & Li, Q. (1994) Biochem. J., 299, 101-107), Signalsequenz der menschlichen plazentalen alkalischen Phosphatase (Mroczkowski, B.S. et al. (1994), J. Biol. Chem., 269, 13522 - 28), Signalsequenz von Melittin von der Honigbiene (Mroczkowski, B.S. et al. (1994), J. Biol. Chem., 269, 13522 - 28; Chai, H. et al. (1993) Biotechnol. Appl. Biochem. (1993) 18, 259 - 73), Signalsequenz des menschlichen Plasminogen- Aktivators (Jarvis, D.L & Summers, M.D. (1989) Mol. Cell. Biol., 9, 214 - 23), Signalsequenzen bestimmter Insekteπzellproteine (WO90/05783) oder Leadersequenzen prokaryotischer Gene (EP-A1 -0 486 170). Ein anderer Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Herstellung von rekombinanten Insektenviren, die für ein Protein aus der DEAD- Proteinsuperfamilie gemäß der vorliegenden Erfindung kodieren, bei dem ein erfindungsgemäßer Vektor zusammen mit Insektenvirus-Wildtyp-DNA in Insektenzellen eingebracht wird und die entstandenen rekombinanten Insektenviren isoliert werden.In a further preferred embodiment, the 5 ′ region of the nucleic acid according to the invention contains a nucleic acid which is suitable for a signal sequence, for example an insulin signal sequence, for example according to SEQ ID No. 13 in the form of the construct p135-NT5S, coded. This construct also has the advantage that the desired protein can be worked up and purified particularly easily, since it is secreted directly into the culture medium due to the signal sequence and the signal sequence is thereby split off instead of accumulating the desired protein intracellularly in the insect cells. Other suitable signal sequences are the signal sequence of silk worm bombyxin (Congote, LF & Li, Q. (1994) Biochem. J., 299, 101-107), signal sequence of human placental alkaline phosphatase (Mroczkowski, BS et al. (1994) , J. Biol. Chem., 269, 13522-28), signal sequence of melittin from the honeybee (Mroczkowski, BS et al. (1994), J. Biol. Chem., 269, 13522-28; Chai, H. et al. (1993) Biotechnol. Appl. Biochem. (1993) 18, 259-73), signal sequence of the human plasminogen activator (Jarvis, DL & Summers, MD (1989) Mol. Cell. Biol., 9, 214-23 ), Signal sequences of certain insect cell proteins (WO90 / 05783) or leader sequences of prokaryotic genes (EP-A1-0 486 170). Another object of the present invention is a method for producing recombinant insect viruses which code for a protein from the DEAD protein superfamily according to the present invention, in which a vector according to the invention is introduced into insect cells together with insect virus wild-type DNA, and the resulting recombinants Insect viruses to be isolated.
Ein geeignetes Insektenvirus ist beispielsweise das Baculovirus, insbesondere das Autographa Californica-Virus. Als Insektenzellen sind beispielsweise Spodoptera Frugiperda, Trichoplusia ni, Rachiplusia ou oder Galleria Mellonela geeignet. Insbesondere geeignet sind die Autographa Californica-Stämme E2, R9, S1 oder S3, vor allem der Autographa Californica-Stamm S3, Spodoptera Frugiperda Stamm 21 oder Trichoplusia ni Eizellen. Neben den Insekteπzellen eignen sich auch Ovaπenzellen der entsprechenden Insekten bzw. deren Larven. Das erfindungsgemäße rekombinante Insektenvirus entsteht in den Iπsektenzellen durch homologe Rekombination des erfindungsgemäßen Vektors mit dem betreffenden Insektenvirus-Wildtyp (siehe z.B. EP-B1-0127839 oder U.S. Pat. 5,004,687). Das rekombinante Insektenvirus kann anschließend zur Herstellung des gewünschten Proteins verwendet werden.A suitable insect virus is, for example, the baculovirus, in particular the Autographa Californica virus. Examples of suitable insect cells are Spodoptera Frugiperda, Trichoplusia ni, Rachiplusia ou or Galleria Mellonela. The Autographa Californica strains E2, R9, S1 or S3 are particularly suitable, especially the Autographa Californica strain S3, Spodoptera Frugiperda strain 21 or Trichoplusia ni egg cells. In addition to the insect cells, oval cells of the corresponding insects or their larvae are also suitable. The recombinant insect virus according to the invention arises in the insect cells by homologous recombination of the vector according to the invention with the wild-type insect virus in question (see, for example, EP-B1-0127839 or U.S. Pat. 5,004,687). The recombinant insect virus can then be used to produce the desired protein.
Ein weiterer Gegenstand der vorliegenden Erfindung bezieht sich daher auch auf ein Verfahren zur Herstellung eines Proteins aus der DEAD-Proteinsuperfamilie, bei dem ein erfindungsgemäßer Vektor oder ein erfindungsgemäßer rekombinanter Insektenvirus in Insektenzellen oder Insektenlarven eingebracht wird, die Insektenzellen bzw. -larven unter geeigneten Bedingungen kultiviert werden und das exprimierte Protein isoliert wird. Vorzugsweise werden Insekteπzellen mit rekombinantem Insektenvirus infiziert, wobei die Infektionsdauer vorzugsweise ca. 40 bis ca. 90, insbesondere ca. 70 Stunden beträgt. Die Herstellung eines rekombinanten Insektenvirus bzw. die Herstellung eines gewünschten Proteins in Insektenzellen erfolgt nach dem Fachmann allgemein bekannten Verfahren, wie sie z.B. in EP-B1-0127839 oder U.S. Pat. 5,004,687 beschrieben sind. Es eignen sich jedoch auch kommerziell erhältliche Baculovirus Expressionssysteme wie z.B. der Baculo Gold™ Transfection kit von Pharmingen oder das Bac-to-Bac™ Baculovirus Expressionsystems von Gibco BRL.Another object of the present invention therefore also relates to a method for producing a protein from the DEAD protein superfamily, in which a vector or a recombinant insect virus according to the invention is introduced into insect cells or insect larvae, which cultivates the insect cells or larvae under suitable conditions and the expressed protein is isolated. Insect cells are preferably infected with recombinant insect virus, the duration of infection preferably being about 40 to about 90, in particular about 70 hours. The production of a recombinant insect virus or the production of a desired protein in insect cells is carried out by methods which are generally known to the person skilled in the art, such as are described, for example, in EP-B1-0127839 or US Pat. No. 5,004,687. It is suitable however, also commercially available baculovirus expression systems such as, for example, the Baculo Gold ™ transfection kit from Pharmingen or the Bac-to-Bac ™ baculovirus expression system from Gibco BRL.
Ein Vorteil der erfindungsgemäßen Insektenzellexpressionsvektoren bzw. der erfindungsgemäßen Verfahren ist es, daß überraschenderweise größere Mengen, im allgemeinen ca. 300-400 mg pro 109 Zellen, an Proteinen aus der DEAD- Proteinsuperfamilie, insbesondere von Proteinen mit einer Molekularmasse von > ca. 100 kD und vor allem Proteine mit einer sogenannten SR-Domäne, hergestellt werden können.An advantage of the insect cell expression vectors according to the invention or the methods according to the invention is that, surprisingly, larger amounts, generally about 300-400 mg per 10 9 cells, of proteins from the DEAD protein superfamily, in particular of proteins with a molecular mass of> about 100 kD and especially proteins with a so-called SR domain can be produced.
Ein weiterer Gegenstand der vorliegenden Erfindung bezieht sich daher auf die Verwendung eines erfindungsgemäßeπ Insektenzell-Vektors zur Herstellung eines Proteins aus der DEAD-Proteinsuperfamilie. Die bezeichneten Proteine eignen sich beispielsweise zur Herstellung entsprechender Testsysteme gemäß EP-A-0778347 oder zur Behandlung einer Erkrankung wie in EP-A-0778347 oder bei Ellis N.A. (1997), supra, beschrieben.Another object of the present invention therefore relates to the use of an insect cell vector according to the invention for producing a protein from the DEAD protein superfamily. The designated proteins are suitable, for example, for the production of corresponding test systems according to EP-A-0778347 or for the treatment of a disease as in EP-A-0778347 or in Ellis N.A. (1997), supra.
Die folgenden Figuren und Beispiele sollen die Erfindung näher erläutern, ohne sie darauf zu beschränken.The following figures and examples are intended to explain the invention in more detail without restricting it thereto.
BESCHREIBUNG DER FIGUREN UND SEQUENZENDESCRIPTION OF THE FIGURES AND SEQUENCES
Figur 1 zeigt schematisch die konservierten Bereiche der Proteine aus der DEAD- Proteinsuperfamilie und der DEAH- und DEXH-Unterfamilien, sowie als Beispiel die konservierten Bereiche des Proteins elF-4A. Die Nummern zwischen den Bereichen geben die Abstände in Aminosäuren an. X steht für eine beliebige Aminosäure.FIG. 1 shows schematically the conserved areas of the proteins from the DEAD protein superfamily and the DEAH and DEXH subfamilies, and, as an example, the conserved areas of the protein elF-4A. The numbers between the areas indicate the distances in amino acids. X stands for any amino acid.
Figur 2 beschreibt schematisch die konservierten Bereiche und deren bekannte Funktionen der Proteine zu der DEAD-, DEAH-, DEXH- bzw. DEAH*-Familien, nach Fuller-Pace, F.V (1994), supra. SEQ ID No. 7 gibt die Aminosäuresequenz der humanen RNA-Helicase p135 wieder. Die RS-Domäne ist die Position 131 bis 253.FIG. 2 schematically describes the conserved areas and their known functions of the proteins for the DEAD, DEAH, DEXH and DEAH * families, according to Fuller-Pace, FV (1994), supra. SEQ ID No. 7 shows the amino acid sequence of the human RNA helicase p135. The RS domain is position 131 to 253.
SEQ ID No. 8 gibt die Nukleiπsäuresequenz der humanen RNA-Helicase p135 wieder.SEQ ID No. 8 shows the nucleic acid sequence of the human RNA helicase p135.
SEQ ID No. 9 gibt die Nukleinsäuresequenz der humanen RNA-Helicase p135 einschließlich deren 3'-nicht-kodierenden Bereich wieder.SEQ ID No. 9 shows the nucleic acid sequence of the human RNA helicase p135 including its 3 'non-coding region.
SEQ ID No. 10 ist p135-NT3 SEQ ID No. 11 ist p135-Pi3 SEQ ID No. 12 ist p135-NT5C SEQ ID No. 13 ist p135-NT5S SEQ ID No. 14 ist p135-NTPS SEQ ID No. 15 ist p135-NTGEX.SEQ ID No. 10 is p135-NT3 SEQ ID No. 11 is p135-Pi3 SEQ ID No. 12 is p135-NT5C SEQ ID No. 13 is p135-NT5S SEQ ID No. 14 is p135-NTPS SEQ ID No. 15 is p135-NTGEX.
Figuren 3 A und B zeigen schematisch den pAcHLT-A Baculovirus-Transfervektor und kodierende Sequenzen des Fremdanteils im Fusionsprotein (Invitrogen ®). 3 B entspricht SEQ ID No. 16 und SEQ ID No. 17FIGS. 3A and B schematically show the pAcHLT-A baculovirus transfer vector and coding sequences of the foreign component in the fusion protein (Invitrogen®). 3 B corresponds to SEQ ID No. 16 and SEQ ID No. 17
Figuren 4 A und B zeigen schematisch den pFASTBACI Baculovirus-Transfervektor und die Klonierungsstelle (Gibco-BRL). 4 B entspricht SEQ ID No. 18.Figures 4 A and B schematically show the pFASTBACI baculovirus transfer vector and the cloning site (Gibco-BRL). 4 B corresponds to SEQ ID No. 18th
Figur 5 zeigt schematisch die Herstellung des Vektors KL33. p135-CDS bedeutet die kodierende p135-DNA-Sequeπz vom 2. kodierenden Baseπtriplett bis zum letzten kodierenden Basentriplett. p135-GS bedeutet die kodierende p135-DNA- Sequenz vom 2. kodierenden Basentriplett bis zur letzten Base des 3'- nichttranslatierten Bereiches. Figur 6 zeigt einen Überblick über die eingesetzten Genkonstrukte zur Expression des p135-Proteins in verschiedenen Wirtszellen.Figure 5 shows schematically the production of the vector KL33. p135-CDS means the coding p135-DNA sequence from the 2nd coding base triplet to the last coding base triplet. p135-GS means the coding p135-DNA sequence from the 2nd coding base triplet to the last base of the 3'-untranslated region. FIG. 6 shows an overview of the gene constructs used for the expression of the p135 protein in different host cells.
Figur 7 zeigt eine Übersicht über Deletionskonstrukte von p135.FIG. 7 shows an overview of deletion constructs of p135.
BeispieleExamples
Beispiel 1example 1
Herstellung eines rekombinanten Baculovirus-Expressionsvektors für die cytosolische Expression des p135 ProteinsProduction of a recombinant baculovirus expression vector for the cytosolic expression of the p135 protein
Alle rekombinanten DNA Methoden wurden anhand von Staπdardmethoden durchgeführt (siehe z. B. Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press).All recombinant DNA methods were carried out using standard methods (see, for example, Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual 2nd ed. Cold Spring Harbor Laboratory Press).
Zur Klonierung der Plasmide (siehe auch Klonierungsschema gemäß Fig. 5) wurde der E.coli-Stamm TOP10 (Invitrogen®) eingesetzt. Zum exakten Einpassen der DNA, die für das p135-Protein aus EP-A-0 778 347 kodiert, in den Vektor pAcHLT-A (Invitrogen®, siehe Fig. 8A) wurde der N-Terminus über PCR amplifiziert. Dazu wurden die OligodesoxynukleotideFor the cloning of plasmids (see cloning scheme of FIG. 5) was the E. coli strain TOP10 (Invitrogen ®) are used. For precise fitting of the DNA which encodes the p135 protein from EP-A-0778347, in the vector pAcHLT-A (Invitrogen ®, see FIG. 8A) of the N-terminus was amplified via PCR. For this purpose, the oligodeoxynucleotides
N1 : 5'-ATGAATTCGGGGACACCAGTGAGGATGCCTCG-3' (SEQ ID No. 3) und N2: 5'-CCGATAATGTCTGTCTTTCCGGATATT-3' (SEQ ID No. 4)N1: 5 ' -ATGAATTCGGGGACACCAGTGAGGATGCCTCG-3 ' (SEQ ID No. 3) and N2: 5 ' -CCGATAATGTCTGTCTTTCCGGATATT-3 ' (SEQ ID No. 4)
verwendet. Nach Restriktionsspaltung mit EcoRI und BspEI wurde das PCR- Fragment zusammen mit dem BspEI-Notl-Fragment der cDNA des p135-Proteins in eine Ligationsreaktion mit dem Vektor pAcHLT-A, welcher mit den Enzymen EcoRI und Notl linearisiert wurde, eingesetzt. Das so erhaltene Plasmid KL33 wurde durch DNA-Sequenzierung bestätigt. In dem so erhaltenen Vektor KL33 ist der 5'- nichttranslatierte Bereich der p135 cDNA durch einen sogenannten Hexahistidin- Tag ersetzt. Dieser Sequenzabschnitt kodiert eine Abfolge von 6 Histidinresten, die den Nachweis sowie die Reinigung des erhaltenen Fusionsproteins erleichtern. In dem erhaltenen Vektor ist zudem zwischen dem Stopp-Codon der p135 cDNA und die vom Vektor pAcHLT-A vorgegebene Terminatorsequenz ein kurzer Abschnitt der 3'-nicht-kodierenden DNA eingesetzt.used. After restriction cleavage with EcoRI and BspEI, the PCR fragment together with the BspEI-Notl fragment of the cDNA of the p135 protein was used in a ligation reaction with the vector pAcHLT-A, which was linearized with the enzymes EcoRI and Notl. The plasmid KL33 thus obtained was by DNA sequencing confirmed. In the vector KL33 obtained in this way, the 5'-untranslated region of the p135 cDNA is replaced by a so-called hexahistidine tag. This sequence section encodes a sequence of 6 histidine residues which facilitate the detection and the purification of the fusion protein obtained. In the vector obtained, a short section of the 3 'non-coding DNA is also inserted between the stop codon of the p135 cDNA and the terminator sequence specified by the vector pAcHLT-A.
Im nächsten Schritt wurde das Plasmid KL33 in eine Baculo-Virus-Kotransfektion eingesetzt. Dazu wurden 1x106 Spodoptera frugiperda Ovarienzellen SF21 -Zellen (Invitrogen®) mit 1 μg Baculo-Gold-DNA (Pharmingen®) und 2 μg KL33 in 70 μl serumfreien Medium mit 30 μl Lipofectin (Invitrogen®) transformiert.In the next step, the plasmid KL33 was used in a baculovirus co-transfection. To this was 1x10 6 Spodoptera frugiperda ovary cells SF21 cells (Invitrogen ®) with 1 ug Baculo Gold DNA (Pharmingen ®) and 2 ug KL33 ul serum-free medium in 70 to 30 ul Lipofectin (Invitrogen ®) transformed.
Zur Identifizierung rekombinanter Baculoviren wurde anschließend ein Plaque-Test durchgeführt. So isolierte PIaques wurden dann mit 1x106 SF21 -Zellen (Invitrogen®) inkubiert. Anschließend wurde die Virus-DNA isoliert und als Matrize für eine PCR eingesetzt. Bei dieser Test-PCR wurden die Oligodesoxynukleotide N1 und N2 verwendet. Nur bei rekombinanten Baculoviren nicht aber bei Wildtyp-Baculoviren zeigte sich bei Analyse der PCR-Ansätze im Agarosegel eine Bande von ca. 310 bp. Zur weiteren Bestätigung wurde der Klon KL33 sequenziert.A plaque test was then carried out to identify recombinant baculoviruses. Thus isolated plaques were then incubated with 1x10 6 SF21 cells (Invitrogen ®). The virus DNA was then isolated and used as a template for a PCR. The oligodeoxynucleotides N1 and N2 were used in this test PCR. Only in the case of recombinant baculoviruses, but not in the case of wild-type baculoviruses, a band of approx. 310 bp was found when analyzing the PCR batches in the agarose gel. Clone KL33 was sequenced for further confirmation.
Mit 200 μl rekombinanten Viren wurden in 75 cm2 Gewebekulturflachen gut gewachsene SF21 -Kulturen (ca. 2 x 107 Zellen) infiziert und 7 Tage bei 27°C inkubiert, um ausreichenden BV33-Stock für die nachfolgenden Proteinexpressionen in Trichoplusia ni Eizellen („High-Five-Zellen", Invitrogen®) zu erhalten. Zur weiteren Vermehrung wurden 3ml dieser Stocklösung in 100 ml SF21 - Kultur (* 2 x 108 Zellen) in 250 ml Spinner (Technomara ®) 7 Tage bei 27 °C inkubiert. Der Virustiter der BV33-Stocklösung wurde mit dem Virustiter-Assay bestimmt. Zur Proteinexpression wurden 100 ml HF-Zellen in 1 I Rollerflaschen bei einer Zelldichte von 2 x 106 /ml mit einer m.o.i. („multiplicity of infection") von 3 mit BV33 Virusstock infiziert. Mit den infizierten High-Five-Zellen wurden Expressionsstudien durchgeführt, um die optimale Inkubationsdauer nach Infektion zu ermitteln. Getestet wurde dabei die Ausbeute an rekombinaπtem p135 anhand der Bandeniπtensität im SDS-Gel. Nach der entsprechenden Inkubationsdauer wurden die Zellen durch Zentrifugation bei 1.500 g pelletiert und bis zur apparenten Homogenität gereinigt, d.h. es konnte nur eine Proteinbande visuell detektiert werden. Hierbei stellte sich eine Infektionsdauer von ca. 72 Stunden als optimal heraus. Nach Aufschluß der Zellen durch Passagiere im Homogenisator wurden die Proteine in 8 M Harnstoff gelöst. Das gewünschte p135-Protein wurde aus dieser Lösung bis zur Homogenität aufgearbeitet. Aus 1x109 „High-Five-Zellen" konnten so ca. 360 mg Protein gewonnen werden.Well-grown SF21 cultures (approx. 2 × 10 7 cells) were infected with 200 μl of recombinant viruses in 75 cm 2 tissue culture areas and incubated for 7 days at 27 ° C. in order to obtain sufficient BV33 stock for the subsequent protein expressions in Trichoplusia ni egg cells (“ High-Five-Cells ", Invitrogen ® ). 3 ml of this stock solution in 100 ml SF21 culture (* 2 × 10 8 cells) in 250 ml spinner (Technomara ®) were incubated for 7 days at 27 ° C. for further propagation. The virus titer of the BV33 stock solution was determined using the virus titer assay. For protein expression, 100 ml of HF cells in 1 liter roller bottles at a cell density of 2 × 10 6 / ml with a moi (“multiplicity of infection”) of 3 were infected with BV33 virus stock. Expression studies were carried out with the infected high-five cells The yield of recombinant p135 was tested on the basis of the band intensity in the SDS gel.After the corresponding incubation period, the cells were pelleted by centrifugation at 1,500 g and cleaned to apparent homogeneity, ie it could only A protein band was detected visually, and an infection duration of about 72 hours was found to be optimal. After disruption of the cells by passengers in the homogenizer, the proteins were dissolved in 8 M urea. The desired p135 protein was worked up from this solution to homogeneity Approx. 360 mg protein could be obtained from 1x10 9 "high five cells".
Beispiel 2Example 2
Herstellung eines rekombinanten Baculovirus-Expressionsvektors für die sekretorische Expression des p135 ProteinsProduction of a recombinant baculovirus expression vector for the secretory expression of the p135 protein
Für die sekretorische Expression des p135 Proteins wurde die p135-DNA in den Vektor pFASTBACI (Gibco-BRL) kloniert (siehe Fig. 4A und B). Über die BamHi und EcoRI-Schnittstelle dieses Vektors wurde zunächst die aus der Hybridisierung synthetischer Oligodesoxynukleotide und anschließende Fill-in-Synthese gewonnene Insulinsignalsequenz (p135-NT5S, SEQ ID No. 12) einkloniert. Anschließend wurde der so erhaltene Vektor FB1 durch Restriktion mit EcoRI und Notl linearisiert. In einem Ligationsansatz wurde der so linearisierte Vektor zusammen mit der Sequenz p135-GS aus dem Vektor KL33 in Anwesenheit von T4- Ligase inkubiert. Mit 1 ng des so erhaltenen Klons FB2 wurden 100 μl kompetente E.coli DH10Bac-Zellen (Gibco-BRL) transformiert. Rekombinante Bacmide lassen sich dabei aufgrund ihrer Weißfärbung beim Blue- White-Screening identifizieren. Weiße Kolonien wurden auf frischen Platten ausgestrichen, um den Genotyp zu bestätigen. Anschließend wurde in Mini-Lysen nach Angaben des Herstellers (Gibco-BRL) die rekombinante Bacmid-DNA isoliert. Analog zu Beispiel 1 wurde über PCR mit den Oligodesoxynukleotiden N1 und N2 bestätigt, daß die rekombinanten Bacmide die p135-DNA enthalten. Zur weiteren Bestätigung wurde der Klon BM33 sequenziert.For the secretory expression of the p135 protein, the p135 DNA was cloned into the vector pFASTBACI (Gibco-BRL) (see FIGS. 4A and B). The insulin signal sequence (p135-NT5S, SEQ ID No. 12) obtained from the hybridization of synthetic oligodeoxynucleotides and subsequent fill-in synthesis was first cloned in via the BamHi and EcoRI interface of this vector. The vector FB1 thus obtained was then linearized by restriction with EcoRI and NotI. In a ligation batch, the vector thus linearized was incubated together with the sequence p135-GS from the vector KL33 in the presence of T4 ligase. 100 μl of competent E.coli DH10Bac cells (Gibco-BRL) were transformed with 1 ng of the clone FB2 obtained in this way. Recombinant bacmids can be identified on the basis of their white color in blue-white screening. White colonies were streaked on fresh plates to confirm the genotype. The recombinant bacmid DNA was then isolated in mini-lyses according to the manufacturer (Gibco-BRL). Analogously to Example 1, it was confirmed by PCR with the oligodeoxynucleotides N1 and N2 that the recombinant bacmids contain the p135 DNA. Clone BM33 was sequenced for further confirmation.
Mit 5 μl BM33 und 6 μl Gibco-BRL CellFECTIN™-Reagenz des rekombinanten Bacmids BM33 wurden 9 x 105 SF21 -Zellen transformiert und 60 h bei 27°C inkubiert. Die Vermehrung der rekombinanten Viren erfolgte wie unter Beispiel 1 beschrieben.9 × 10 5 SF21 cells were transformed with 5 μl BM33 and 6 μl Gibco-BRL CellFECTIN ™ reagent of the recombinant bacmid BM33 and incubated at 27 ° C. for 60 h. The recombinant viruses were propagated as described in Example 1.
Das Protein p135 wurde nach 72 h aus den Überständen infizierter HF-Zellen (angezogen in einem Medium wie in Beispiel 1 beschrieben, allerdings ohne FCS) durch Konzentrierung des Überstandes und anschließende chromatographische Reinigung in appareπter Homogenität erhalten. Durch die Sekretion ins Kulturmedium war die Aufarbeitung und Reinigung wesentlich erleichtert.After 72 h, the protein p135 was obtained from the supernatants of infected HF cells (grown in a medium as described in Example 1, but without FCS) by concentrating the supernatant and subsequent chromatographic purification with appropriate homogeneity. The secretion into the culture medium made the processing and cleaning considerably easier.
Vergleichsbeispiel 1Comparative Example 1
Expressionsversuche mit dem Bakterium E.coiiExpression experiments with the E.coii bacterium
Zur Expression in E.coii wurden zwei Full-Length-Konstrukte von p135-DNA (siehe Fig. 11 ) mit unterschiedlichem C-Terminus in den Vektor pGEX-4T-1 (Pharmacia®) einkloniert. Die Klonierung erfolgte über die Restritkionsschnittstellen EcoRI und Notl des Vektors. Die Fragmente wurden direkt aus den Baculovektoren umkloniert. Konstrukt Ec33-M enthält p135-DNA ohne die native 3'-nichttraπslatierte Sequenz. Um dieses Konstrukt zu erhalten, wurde eine PCR mit den Oligodesoxynukleotiden T1: 5'-TGA CAG TCG GAC TTA GTC CTAACG CCG GCG ATATGC ATG C-3' For expression in E.coii two full-length constructs were cloned from p135 DNA (see Fig. 11) with different C-terminus into the vector pGEX-4T-1 (Pharmacia ®). The cloning was carried out via the restriction sites EcoRI and Notl of the vector. The fragments were cloned directly from the baculo vectors. Construct Ec33-M contains p135-DNA without the native 3 ' non-translated sequence. In order to obtain this construct, a PCR with the oligodeoxynucleotides was carried out T1: 5'-TGA CAG TCG GAC TTA GTC CTAACG CCG GCG ATATGC ATG C-3 '
(SEQ ID No.5) und T2: 5'-GCC TCT GCC ATG GAG GAG GAG ATG-3' (SEQ ID No.6)(SEQ ID No.5) and T2: 5'-GCC TCT GCC ATG GAG GAG GAG ATG-3 ' (SEQ ID No.6)
durchgeführt. Dieses Fragment wurde nach Restπktion mit Ncol und Notl gegen den nativen C-Terminus von p135 ausgetauscht.carried out. After fragmentation with Ncol and Notl, this fragment was exchanged for the native C-terminus of p135.
Mit den beiden Klonen Ec33-N und Ec33-M wurden E.coii TOP10 Zellen (Invitrogen®) transformiert. Die erhaltenen transformierten Zellen wurden nach Bestätigung der erfolgreichen Transformation über Minilysen zu einer OD800 von 0,7 angezogen und dann mit IPTG induziert. Nach 1 , 2, 3 und 4 Stunden wurden Aliquots im SDS-PAGE auf Expression hin analysiert. Es konnten keine Expressionsprodukte nachgewiesen werden.E.coii TOP10 cells (Invitrogen ® ) were transformed with the two clones Ec33-N and Ec33-M. The transformed cells obtained were, after confirmation of the successful transformation, minilysed to an OD800 of 0.7 and then induced with IPTG. After 1, 2, 3 and 4 hours, aliquots were analyzed for expression in SDS-PAGE. No expression products could be detected.
Zur Überprüfung, daß das Expressionssystem korrekt funktionierte, wurden gemäß obiger Beschreibung die Deletionskonstrukte D1 und D2 exprimiert (siehe Fig. 7). Mit dem Konstrukt D2, das ein internes Fragment darstellt, konnten Expressionsprodukte nachgewiesen werden.To check that the expression system was functioning correctly, the deletion constructs D1 and D2 were expressed as described above (see FIG. 7). Expression products could be detected with construct D2, which represents an internal fragment.
Vergleichsbeispiel 2Comparative Example 2
Expressionsversuche mit der Hefe Pichia pastorisExpression experiments with the yeast Pichia pastoris
Zur Expression in P. pastoris wurden zwei verschiedene Full-Length-Konstrukte in die Pichia-Vektoren pICZDA bzw. pICZB (Invitrogen®, siehe auch http://www.invitrogen.com) kloniert. Die Konstrukte (siehe Fig. 6) wurden durch Restnktionsverdau aus den E.coli-Konstrukten Ec33-N und Ec33-M isoliert und über die Schnittstellen EcoRI und Notl in die Pichia-Vektoren eingebaut.For expression in P. pastoris, two different full-length constructs were cloned into the Pichia vectors pICZDA or pICZB (Invitrogen ®, see also http://www.invitrogen.com). The constructs (see FIG. 6) were isolated from the E. coli constructs Ec33-N and Ec33-M by residual digestion and incorporated into the Pichia vectors via the interfaces EcoRI and Notl.
Transformation der P. pastoris-Zellen vom Stamm KM71 erfolgte gemäß den Angaben des Herstellers (Invitrogen®). Ebenso wurde für die Analyse der Proteinexpression nach Angaben des Herstellers (Invitrogen®, Nr. K1740-01) verfahren. Es konnten keine Expressionsprodukte nachgewiesen werden.The P. pastoris cells from the KM71 strain were transformed according to the manufacturer's instructions (Invitrogen ® ). Likewise, for the analysis of the Proceed protein expression according to the manufacturer (Invitrogen ® , No. K1740-01). No expression products could be detected.
Zur Überprüfung, daß das Expressionssystem korrekt funktionierte, wurden gemäß obiger Beschreibung die Deletionsklone D1 und D2 erfolgreich exprimiert. To check that the expression system was functioning correctly, the deletion clones D1 and D2 were successfully expressed as described above.

Claims

Patentansprüche claims
1. Insektenzell-Vektor enthaltend eine Nukleinsäure kodierend für ein Protein aus der DEAD-Proteinsuperfamilie.1. Insect cell vector containing a nucleic acid coding for a protein from the DEAD protein superfamily.
2. Vektor nach Anspruch 1 , dadurch gekennzeichnet, daß die genannte Nukleinsäure am 3'-Ende des kodierenden Bereiches einen nativen 3'-nicht- kodierenden Bereich enthält.2. Vector according to claim 1, characterized in that said nucleic acid at the 3'-end of the coding region contains a native 3'-non-coding region.
3. Vektor nach Anspruch 2, dadurch gekennzeichnet, daß der 3'-nicht-kodierende Bereich mindestens ca. 50, vorzugsweise ca. 50 bis ca. 450, insbesondere ca. 50 bis ca. 400 Nukleotide lang ist.3. Vector according to claim 2, characterized in that the 3'-non-coding region is at least approximately 50, preferably approximately 50 to approximately 450, in particular approximately 50 to approximately 400 nucleotides long.
4. Vektor nach Anspruch 2 oder 3, dadurch gekennzeichnet, daß der 3'-nicht- kodierende Bereich eine Bindestelle für das CPSF-Protein, CstF-Protein, CF I- Protein, CF Il-Protein, Poly(A)-Polymerase und/oder Poly(A)-bindende Protein II (PAB II) enthält.4. Vector according to claim 2 or 3, characterized in that the 3'-non-coding region is a binding site for the CPSF protein, CstF protein, CF I protein, CF II protein, poly (A) polymerase and / or contains poly (A) -binding protein II (PAB II).
5. Vektor nach einem der Ansprüche 2-4, dadurch gekennzeichnet, daß der 3'- nicht-kodierende Bereich eine AATAAA-Bindestelle, eine ATTAAA-Bindestelle, ein GT-reiches Element, insbesondere ein YGTGTTYY-Elemeπt, und/oder ein T-reiches Element enthält.5. Vector according to one of claims 2-4, characterized in that the 3'- non-coding region is an AATAAA binding site, an ATTAAA binding site, a GT-rich element, in particular a YGTGTTYY element, and / or a T. - Contains rich element.
6. Vektor nach einem der Ansprüche 1 -5, dadurch gekennzeichnet, daß die Nukleinsäure für ein Protein aus der DEAD-, DEAH-, DEXH und/oder DEAH*- Proteinfamilie kodiert.6. Vector according to one of claims 1 -5, characterized in that the nucleic acid codes for a protein from the DEAD, DEAH, DEXH and / or DEAH * protein family.
7. Vektor nach einem der Ansprüche 1 -6, dadurch gekennzeichnet, daß die Nukleinsäure für ein Protein mit Nukleinsäure-bindender Aktivität, Helicase- Aktivität und/oder ATPase-Aktivität kodiert. 7. Vector according to any one of claims 1-6, characterized in that the nucleic acid codes for a protein with nucleic acid binding activity, helicase activity and / or ATPase activity.
8. Vektor nach einem der Ansprüche 1-7, dadurch gekennzeichnet, daß die Nukleinsäure für ein Protein kodiert, welches Zellen Toleranz gegenüber Isoxazolderivaten und wirkungsverwandten Verbindungen verleiht.8. Vector according to one of claims 1-7, characterized in that the nucleic acid codes for a protein which gives cells tolerance to isoxazole derivatives and related compounds.
9. Vektor nach einem der Ansprüche 1-8, dadurch gekennzeichnet, daß die Nukleinsäure für ein humanes Protein kodiert.9. Vector according to one of claims 1-8, characterized in that the nucleic acid codes for a human protein.
10. Vektor nach einem der Ansprüche 1-9, dadurch gekennzeichnet, daß die Nukleinsäure für ein Protein mit einer Sequenz gemäß SEQ ID No. 7 kodiert, wobei die Figur Teil des Anspruchs ist.10. Vector according to one of claims 1-9, characterized in that the nucleic acid for a protein with a sequence according to SEQ ID No. 7 encodes, the figure being part of the claim.
11. Vektor nach einem der Ansprüche 1-10, dadurch gekennzeichnet, daß die Nukleinsäure die Sequenz gemäß SEQ ID No. 8 enthält, wobei die Figur Teil des Anspruchs ist.11. Vector according to one of claims 1-10, characterized in that the nucleic acid has the sequence according to SEQ ID No. 8 contains, the figure being part of the claim.
12. Vektor nach einem der Ansprüche 1-11 , dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure Insektenzell-spezifische regulatorische Nukleotidsequenzen vorhanden sind.12. Vector according to one of claims 1-11, characterized in that insect cell-specific regulatory nucleotide sequences are present at the 5 'end of the nucleic acid mentioned.
13. Vektor nach einem der Ansprüche 1-12, dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure ein Polyhedrin-Promoter enthalten ist.13. Vector according to one of claims 1-12, characterized in that a polyhedrin promoter is contained at the 5 'end of the nucleic acid mentioned.
14. Vektor nach einem der Ansprüche 1-13, dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure eine Polyhedrin-ATG-Traπslations-Initiations- Startstelle enthalten ist.14. Vector according to any one of claims 1-13, characterized in that a polyhedrin-ATG translation initiation starting point is contained at the 5 'end of said nucleic acid.
15. Vektor nach einem der Ansprüche 1-14, dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure eine Nukleinsäure enthalten ist, die für ein Oligopeptid aus mindestens ca. 4 Histidinen, vorzugsweise aus ca. 6 Histidinen und/oder für die Glutathion-S-transferase kodiert. 15. Vector according to any one of claims 1-14, characterized in that a nucleic acid is contained at the 5 'end of said nucleic acid, which for an oligopeptide from at least about 4 histidines, preferably from about 6 histidines and / or for Encoded glutathione-S-transferase.
16. Vektor nach Anspruch 15, dadurch gekennzeichnet, daß am 5'-Ende der genannten Nukleinsäure eine Nukleinsäure mit der Sequenz gemäß SEQ ID No. 12 enthalten ist, wobei die Figur Bestandteil des Anspruchs ist.16. Vector according to claim 15, characterized in that at the 5 'end of said nucleic acid a nucleic acid with the sequence according to SEQ ID No. 12 is included, the figure being part of the claim.
17. Vektor nach einem der Ansprüche 1-16, dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure eine Nukleinsäure enthalten ist, die für eine Protease-Schnittstelle kodiert.17. Vector according to one of claims 1-16, characterized in that a nucleic acid is present at the 5 'end of said nucleic acid, which codes for a protease interface.
18. Vektor nach Anspruch 17, dadurch gekennzeichnet, daß die zusätzliche Nukleinsäure für eine Thrombin-Schnittstelle mit der Sequenz Leu-Val-Pro-Arg- Gly-Ser (SEQ ID No. 1) und/oder für eine FXa-Schnittstelle mit der Sequenz lle- Glu-Gly-Arg (SEQ ID No. 2) kodiert.18. Vector according to claim 17, characterized in that the additional nucleic acid for a thrombin interface with the sequence Leu-Val-Pro-Arg-Gly-Ser (SEQ ID No. 1) and / or for an FXa interface with the Sequence III-Glu-Gly-Arg (SEQ ID No. 2) encoded.
19. Vektor nach einem der Ansprüche 1-18, dadurch gekennzeichnet, daß am 5'- Ende der genannten Nukleinsäure eine Nukleinsäure enthalten ist, die für eine Signalsequenz kodiert.19. Vector according to one of claims 1-18, characterized in that a nucleic acid is present at the 5 'end of said nucleic acid, which codes for a signal sequence.
20. Vektor nach Anspruch 19, dadurch gekennzeichnet, daß die Signalsequenz ausgewählt ist aus einer Signalsequenz von Insulin, Bombyxin, menschliche plazentale alkalische Phosphatase, Melittin, menschlicher Plasminogen- Aktivator, Insektenzellproteine oder Leadersequenzen prokaryotischer Gene.20. Vector according to claim 19, characterized in that the signal sequence is selected from a signal sequence of insulin, bombyxin, human placental alkaline phosphatase, melittin, human plasminogen activator, insect cell proteins or leader sequences of prokaryotic genes.
21. Vektor nach Anspruch 19 oder 20, dadurch gekennzeichnet, daß die Nukleinsäure kodierend für die Signalsequenz eine Sequenz gemäß SEQ ID No. 13 ist, wobei die Figur Teil des Anspruchs ist.21. Vector according to claim 19 or 20, characterized in that the nucleic acid coding for the signal sequence is a sequence according to SEQ ID No. 13, the figure being part of the claim.
22. Verfahren zur Herstellung von rekombinanten Insektenviren, die für ein Protein aus der DEAD-Proteinsuperfamilie kodieren, dadurch gekennzeichnet, daß ein Vektor gemäß einem der Ansprüche 1-21 zusammen mit Insektenvirus-Wildtyp- DNA in Insektenzellen eingebracht wird und die entstandenen rekombinanten Insektenviren isoliert werden.22. A process for the production of recombinant insect viruses which code for a protein from the DEAD protein superfamily, characterized in that a vector according to any one of claims 1-21 together with insect virus wild-type DNA is introduced into insect cells and the resulting recombinant insect viruses are isolated.
23. Verfahren nach Anspruch 22, dadurch gekennzeichnet, daß die Insektenvirus- Wildtyp-DNA Baculovirus-DNA ist.23. The method according to claim 22, characterized in that the insect virus wild-type DNA is baculovirus DNA.
24. Verfahren zur Herstellung eines Proteins aus der DEAD-Proteinfamilie, dadurch gekennzeichnet, daß ein Vektor nach einem der Ansprüche 1-21 oder ein rekombinanter Insektenvirus hergestellt nach einem Verfahren gemäß Anspruch 22 oder 23 in Insektenzellen oder -larven eingebracht wird, die Insektenzellen oder -larven unter geeigneten Bedingungen kultiviert werden und das exprimierte Protein isoliert wird, vorzugsweise werden die Iπsektenzelleπ oder - larven mit rekombinantem Insektenvirus infiziert.24. A method for producing a protein from the DEAD protein family, characterized in that a vector according to any one of claims 1-21 or a recombinant insect virus produced by a method according to claim 22 or 23 is introduced into insect cells or larvae, the insect cells or larvae are cultivated under suitable conditions and the expressed protein is isolated, preferably the insect cell or larvae are infected with recombinant insect virus.
25. Verfahren nach Anspruch 24, dadurch gekennzeichnet, daß die Infektionsdauer der Insektenzellen ca. 40 bis ca. 90, vorzugsweise ca. 70 Stunden beträgt.25. The method according to claim 24, characterized in that the infection duration of the insect cells is about 40 to about 90, preferably about 70 hours.
26. Verfahren nach einem der Ansprüche 22-25, dadurch gekennzeichnet, daß die Insektenzellen Spodoptera frugiperda-Zellen, vorzugsweise Spodoptera frugiperda Ovarienzellen, sind.26. The method according to any one of claims 22-25, characterized in that the insect cells are Spodoptera frugiperda cells, preferably Spodoptera frugiperda ovarian cells.
27. Verwendung eines Insekten-Vektors gemäß einem der Ansprüche 1-21 zur Herstellung eines Proteins aus der DEAD-Proteinfamilie. 27. Use of an insect vector according to any one of claims 1-21 for the production of a protein from the DEAD protein family.
EP99911658A 1998-02-12 1999-02-09 Expression vector for the production of dead proteins Withdrawn EP1054990A2 (en)

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