WO2000023604A1 - Vecteurs d'expression renfermant des sequences regulatrices de stylonychia lemnae pour l'expression de proteine heterologue dans des protistes eucariotiques, et procede d'identification de telles sequences regulatrices - Google Patents

Vecteurs d'expression renfermant des sequences regulatrices de stylonychia lemnae pour l'expression de proteine heterologue dans des protistes eucariotiques, et procede d'identification de telles sequences regulatrices Download PDF

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WO2000023604A1
WO2000023604A1 PCT/EP1999/007958 EP9907958W WO0023604A1 WO 2000023604 A1 WO2000023604 A1 WO 2000023604A1 EP 9907958 W EP9907958 W EP 9907958W WO 0023604 A1 WO0023604 A1 WO 0023604A1
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expression vector
gene
eukaryotic
nucleic acid
vector according
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PCT/EP1999/007958
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German (de)
English (en)
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Karl-Christian Gallert
Christoph Hüls
Stefan Müllner
Günther STEINBRÜCK
Naduparambil Korah Jacob
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Celanese Ventures Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to expression vectors for the production of proteins in the context of heterologous protein expression in eukaryotic protists, preferably in ciliates, in particular in the hypotrich ciliate Stylonychia lemnae. Furthermore, the invention relates to a method for identifying regulatory sequences with defined properties from eukaryotic protists.
  • biotechnologically usable microorganisms e.g. E.coli, S.cerevisiae, P.pastoris, B.subtilis, etc.
  • heterologous protein expression i.e. the synthesis of proteins in a non-native environment.
  • Such recombinant proteins are essentially produced via three
  • Procedural steps It starts with the cloning of the nucleic acid (DNA) of the desired protein into suitable expression vectors and the amplification of these constructs in an intermediate organism (e.g. E. coli). After transfer of this DNA into the desired host organism (so-called DNA transformation), the transformants are selected and the expression level of the desired protein is determined.
  • DNA nucleic acid
  • a functional expression vector must be able to be replicated stably in the foreign organism. For this either a functional starting point of replication together with control sequences for the segregation is necessary, which allows a stable genetic transfer in addition to the genomic DNA, or an integration of the expression construct into the genomic DNA by means of homologous DNA
  • indicator genes ie genes whose gene products can easily be determined qualitatively or quantitatively using spectroscopic methods.
  • protists especially in ciliates, in sufficient quantities in a functionally heterologous manner.
  • Sequences such as promoters, terminators etc. are not available or are not designed for optimal gene dose / protein expression rates (e.g. efficiency of the origin of replication).
  • the use of vectors that include Carrying fragments derived from rDNA can also lead to undesired gene dose effects. This is because the rate of mRNA for the encoded
  • Protein due to the amplification at the DNA level can only be modified to a limited extent.
  • Gaertig et al. (NAR 22, 1994) a more efficient transformation method for protists.
  • no data on expression level and promoter selection are shown here either. Therefore, no qualitative statement is possible.
  • Patent application DE19626564 describes the basic feasibility of heterologous protein expression in Stylonychia lemnae using the method of gene bombardment to transform protists.
  • Eukaryotic protists have functional and structural features that are not only in the interest of basic cell biological research but are also suitable for biotechnological use on an industrial scale.
  • Protists are mostly unicellular eukaryotes that can be grown in clonal cultures analogously to prokaryotic microorganisms in high cell density with relatively short generation times and can therefore also be used economically and cost-effectively in the industrial process. They are also easily accessible for fermentation.
  • protists, enzymes, structural and membrane proteins and metabolic pathways are very similar to the corresponding processes and structures in multicellular eukaryotes (e.g. humans, animals, plants, and much more) than is the case for prokaryotes, provided that corresponding elements are present in prokaryotes at all.
  • Protists have almost all eukaryotic ones
  • prokaryotes lack, e.g. in the field of DNA replication, transcription and processing, translation, cytoskeleton and membrane structures, endo- and exocytosis processes and much more.
  • inclusion bodies in which the expressed foreign protein is mostly present in the host cell in denatured form.
  • the occurrence of such inclusion bodies depends on the choice of an expression vector.
  • the object of the present invention is therefore to provide an expression vector for eukaryotic protists, which enables the production of proteins in eukaryotic protists, preferably ciliates, particularly preferably Stylonychia lemnae, in large quantities.
  • an expression vector for eukaryotic protists which for this purpose has an optimized genetic control sequence for its function, containing regulatory sequences with the functions promoter, operator, ribosomal binding site and marker gene for selection, origin of replication, termination of transcription, etc. (hereinafter the expression vector according to the invention) ).
  • Another object is to provide a promoter test system for
  • Protists in the sense of this invention are eukaryotic protists.
  • eukaryotic protists come from the realms of algae, fungi and protozoa - despite the fact that some strains within the algae and fungi are organized in multiple cells.
  • eukaryotic protists originate from the Protozoa realm, and here essential features of the eukaryotic protists are particularly pronounced for the invention.
  • the invention makes use of the following essential characteristic features in eukaryotic protists: these have a cell nucleus (in contrast to prokaryotic protists, the bacteria and cyanobacteria); they live most of their development cycle as single cells (in contrast to plants and animals) and they have "unstiffened” cell walls (in contrast to fungi and algae). These characteristics are particularly pronounced for Protozoa. Ciliophora (Ciliata) and in particular Stylonychia lemnae are particularly suitable for the invention.
  • Percolozoa Parabasalia, Euglenozoa, Mycetozoa, Entamoebia, Opalozoa, Dinozoa, Apicomplexa, Ciliophora, Haplosporidia, Paramyxia, Rhizopoda, Reticulosa, Heliozoa, Radiozoa, Amoebozoa, Choanozoa
  • Chytridiomycota Zygomycota, Ascomycota, Basidiomycota
  • Chlorophyta Charophyta, Glaucophyta, Rhodophyta
  • Ciliates which have a so-called core dimorphism, can be seen as a particularly advantageous host cell.
  • the micronucleus mainly has generative functions.
  • meiosis creates haploid gamete nuclei.
  • the gamete nuclei of two conjugation partners can fuse to form zygote nuclei and create a new cell generation with genetically recombined micronucleus genomes.
  • the new generation of zygote cores creates new macronuclei by division.
  • the natural amplification of the DNA macronucleus causes a gene dose effect which advantageously determines the choice of the time of a transformation and favors efficiency.
  • an expression vector is a specially constructed “cloning vector” which, after introduction into a suitable host cell - preferably a eukaryotic protist, preferably a ciliate, particularly preferably Stylonychia lemnae, allows the transcription and translation of the foreign gene cloned into the expression vector and thus allows the stable Passing on to descendants.
  • the expression vector according to the invention contains the control signals required for the expression of genes in cells of eukaryotic protists. These control signals - or synonymously regulatory sequences - are essential for expression vectors, because - as is known - for example bacterial genes mostly cannot be expressed in eukaryotic host cells. Likewise, eukaryotic yeast promoters, for example, are not recognized by mammalian eukaryotic cells. In the sense of this invention, an expression cassette for controlling the (foreign) gene expression is spoken synonymously with regulatory sequences.
  • An object of the present invention is therefore an expression vector (or: vector, plasmid) for eukaryotic protists containing a nucleic acid coding for a desired protein with 5 ' and 3 ' regulative sequences (control sequences).
  • This nucleic acid can code for a human, animal, vegetable or bacterial protein.
  • the capacity of the expression vector is up to approx. 20 kBp, preferably 300-5000 nucleotides.
  • the expression vector according to the invention additionally contains, at the 3'-end of the coding region of the coding nucleic acid for a desired protein, a native 3'-non-coding region (regulative sequence with control signals) which in preferred embodiments is at least approximately 50, preferably approximately 150 to is approximately 500, in particular approximately 200 to approximately 300 nucleotides long. A length of 215 nucleotides according to SEQ ID No. is particularly preferred. 1.
  • the 3 ' -flanking region of the expression vector according to the invention consists of a 3 ' non-coding sequence, including the trailer sequence mentioned, with terminal telomeric sequences.
  • the telomeric sequence is: G4T4G4T4G4
  • This 3 ' flanking region is particularly preferably obtainable from Stylonychia lemnae or those organisms which have "genesized pieces (GSP)" in part or as a whole in the genomic DNA.
  • the expression vector according to the invention additionally contains, at the 5 end of the coding region of the coding nucleic acid for a desired protein, a native 5 'non-coding region (regulative sequence with control signals), which in preferred or preferred ones Embodiments is at least about 50, preferably about 150 to about 500, in particular about 200 to about 300 nucleotides long. A length of 259 nucleotides according to SEQ ID No. is particularly preferred. Second
  • the 5 ' flanking region of the expression vector according to the invention consists of a 5 ' non-coding sequence, including the leader sequence mentioned, with terminal telomeric sequences.
  • the telomeric sequence is: C4A4C4A4C4
  • This 5 ' flanking region according to the invention is particularly preferably obtainable from Stylonychia lemnae or organisms such as these in part or as a whole
  • GSP Gene-sized-pieces
  • nucleic acid is preferably understood to mean single- or double-stranded DNA or RNA, in particular double-stranded DNA.
  • Functional heterologous protein expression in the sense of the present invention means the expression of proteins according to the information of the foreign gene in the expression vector, whereby not only the same amino acid sequence of the native protein is obtained, but also defined by means of eukaryotic post-translational modification (for example phosphorylation, attachment of sugar or fatty acid residues or other derivatization) Amino acid side chains) the activity (eg binding to target molecules, eg metabolites, substrates, proteins, nucleic acids, enzymatic function, etc.) corresponds to the native protein.
  • eukaryotic post-translational modification for example phosphorylation, attachment of sugar or fatty acid residues or other derivatization
  • Amino acid side chains eg binding to target molecules, eg metabolites, substrates, proteins, nucleic acids, enzymatic function, etc.
  • the expression vector according to the invention allows the expression of a protein encoded by the foreign gene as an authentic, biologically active enzyme.
  • the expression vector according to the invention carries the 5 ' and 3 ' control signals according to the invention.
  • the expression of a protein encoded by the cloned-in foreign gene is preferably carried out in the sense of a translation fusion.
  • a hybrid protein or fusion protein is obtained which can be easily detected.
  • detectable fusion proteins are obtained within the scope of this invention, for example by means of "green fluorescence protein (GFP)" or “beta-glucuronidase (GUS)".
  • the desired protein is obtained by cleavage from the fusion protein in a manner known to those skilled in the art.
  • the 5 ' and 3 ' control signals necessary for expression are integrated in the expression vector according to the invention.
  • Expression vector have suitable selective marker genes (Zeocin and / or Neomycin), which gives the transformed host cell a growth advantage. These can be expressed with the desired protein as a fusion protein.
  • GSP gene-sized-pieces
  • the 3 ' flanking region of the gene for terminating the transcription with further information for translation is at the 3 ' end of an mRNA and inclusive a 3 ' untranslated region / sequence (untranslated 3 ' region; "3 ' -UTR") or trailer of the cloned (foreign) gene.
  • the trailer region according to the invention is decisive for the stability of the mRNA and the associated translation processes.
  • the native 3 'and 5' are - flanking regions from the alpha tubulin gene of Stylonychia lemnae after
  • codons base triplets
  • a specific codon-amino acid frequency results in the individual species, with respect to these codons preferably amino acids are obtained (one speaks of codon preference of a species or synonymously "codon usage ").
  • flanking regions according to the invention support the codon preference in eukaryotic protists, preferably ciliates, particularly preferably Stylonychia lemnae, in relation to the cloned-in foreign gene, that is to say the nucleic acid mentioned, which codes for a protein.
  • a eukaryotic protist preferably a ciliate, particularly preferably Stylonychia lemnae.
  • the coding nucleic acid (hereinafter: CDS) of the desired protein is preferably converted into an E. coli using the "two-step PCR" method described in the examples (see also FIG. 1) compatible vector (eg vector pSE1; puC 18 or other vectors or plasmids known to the person skilled in the art).
  • CDS coding nucleic acid
  • FIG. 1 the description in the exemplary embodiments for the amplification of the CDS of the desired protein, PCR primers are used which have a 16 to 21 bp overlap with the 5 ' and 3 ' regulatory sequences according to SEQ ID No. 1 and
  • SEQ ID No. 2 have.
  • telomer primers which are obtained from ciliates, preferably Stylonychia lemnae
  • a DNA fragment can be obtained in this way which has ends with recognition sites for the restriction endonucleases Xbal and Apal and is compatible in Smal linearized forms of E. coli Vector can be incorporated as part of a DNA ligation reaction.
  • the modified compatible vector obtained is preferably introduced into the host organism as part of a co-transformation reaction with the vector pST-neo (SEQ ID No. 5).
  • the vector pST-neo allows by functional
  • neomycin resistance gene in protists, especially Stylonychia lemnae, the selection of successful transformants. Due to the high co-transformation rates (> 75%), the clones are also represented which contain the modified compatible vector parallel to pST-neo. The co-transformations are carried out as described above, with an emphasis on optimized
  • Transformation conditions must be observed in order to keep the number of transformants as high as possible.
  • the successfully transformed cells are then used for protein expression.
  • the co-transformation is carried out instead of the (co) vector pST-neo with the (co) vector pSt-tracer (SEQ ID No. 6).
  • the functional fusion protein of GFP and Zeocin is under the control of the alphal tubulin promoter from Stylonychia lemnae. This enables the selection of successful cotransformants to be determined both by zeocin selection and by determining the GFP fluorescence yield.
  • both the GFP-Zeocin fusion protein and the desired protein are under the control of the alphal - Tubulin promoter are, an optimization of the expression level of the desired protein can be carried out in a rapid process indirectly by optimizing the GFP fluorescence.
  • Eukaryotic protists have the ability to reproduce "sexually". This process, which the Protists call conjugation, requires organisms with different mating types. Sexual reproduction occurs in suitable culture conditions. With the possibility of crossing eukaryotic protists of the same kind but different mating types, the exchange of genetic information and the maintenance of strains that are homozygous in certain characteristics are possible.
  • the coding nucleic acid for a desired protein between the ATG translation initiation start site and the region coding for the desired protein contains a nucleic acid coding for an oligopeptide of at least about 4, preferably of about 6, histidines.
  • a fusion protein is obtained from the desired protein and an N-terminally fused peptide which contains the histidines mentioned.
  • the protein can be purified in a particularly simple and effective manner, for example via a chromatography column containing metal ions, such as, for example, 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, it is also possible to use a nucleic acid which codes for the glutathione S-transferase (Smith, DB & Johnson, KS (1988) Gene, 67, 31-40).
  • the fusion proteins obtained in this way can also be easily purified using affinity chromatography and detected using a colorimetric test or an immunoassay.
  • nucleic acid codes for a protease interface.
  • 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.
  • the factor Xa cleavage site contains, for example, the amino acid sequence Ile-Glu-Gly-Arg.
  • Another object of the present invention therefore also relates to a method for producing a desired protein, in which an expression vector according to the invention is introduced into eukaryotic protists, the eukaryotic protists being cultivated under suitable conditions and the expressed protein being isolated. Eukaryotic protists are preferably transformed with recombinant vectors. A desired protein is produced in eukaryotic protists by the methods which are generally known to those skilled in the art.
  • eukaryotic protist cells preferably ciliates, particularly preferably Stylonychia lemnae
  • ciliates particularly preferably Stylonychia lemnae
  • Expression vector has the characteristic of overexpression in eukaryotic protists. Overexpression in the sense of this invention means that 7-40%. preferably 7-15% of the expressed foreign protein is present in the host cell.
  • Another object of the present invention therefore relates to the
  • an expression vector according to the invention for the production of a desired protein.
  • the expression vector used is suitable for any incorporated foreign gene or heterologous nucleic acid from human, animal, plant or bacterial origin.
  • the first step in heterologous protein expression is the introduction of the expression vector according to the invention into the desired organism.
  • the expression level of the desired protein is determined.
  • the focus is on a high protein production rate.
  • An often decisive factor here is the selection of a suitable promoter.
  • this can also allow the induction of protein expression under the most favorable cellular conditions.
  • Another object of the invention relates to the identification of regulatory elements
  • Inductor in defined concentrations change of physical parameters such as Temperature, pH or salinity, changes in the C or N source or in general in the food supply, and much more) can be specifically activated.
  • Metabolism engineering This means the targeted influencing of metabolic pathways or regulatory networks of an organism. This is e.g. very useful when the biosynthetic pathway for an interesting metabolite is limited by a step or a preliminary step. If there is a regulatory bottleneck or prepress limitation targeted
  • Biological production processes can be optimized by manipulating the exchange of the native for a suitable foreign promoter.
  • biological organisms are expanded in their properties and characteristics (e.g. resistance to one
  • Range of stressors such as heat, cold, fluctuating humidity, pests, and much more or certain desired ingredients, such as vitamins, essential fatty acids, and much more).
  • the methods of recombination technologies also make it essential to place the missing or existing genes under the control of regulatory sequences, which provide an optimal "supply" for the biological organisms (eg crop) with the appropriate hnRNA / mRNA guarantee.
  • GSP Gene Sized Pieces
  • the invention therefore also relates to a promoter test system with the method steps:
  • a regulatory sequence is preferably seen as a promoter.
  • a promoter denotes an enhancer region, that is to say a nucleic acid with promoter activity (regulative activity), which controls the transcription of a gene as a cis-acting regulative sequence of approx. 100 nucleotides.
  • This promoter can also be split within the non-coding region (eg within the leader) or be part of an operon or an internal control region.
  • the strength of the promoter there is generally between strong and weak
  • Promoters distinguished. Some promoters can be regulated in strength, while others, the so-called constitutive promoters, cannot are adjustable.
  • nucleic acids with regulatory activity from eukaryotic protists are identified and isolated, which are characterized by defined properties.
  • the eukaryotic protists are treated with the help of agents / protocols of abiotic (heavy metals etc.) and biotic nature. Agents are primarily to be understood as bioactive substances with a sensitive effect for eukaryotic protists. The protocols are varied accordingly until there are reproducible changes in the mRNA spectrum (control: Northern blot, see there) (e.g. cold shock program etc.).
  • the promoter activity of unknown nucleic acid is tested in the context of known promoter test vectors. These vectors carry a promoterless gene whose gene product is easily detectable, but is not expressed due to the lack of a promoter. By inserting a nucleic acid with promoter properties in front of the coding region of the reporter gene (indicator gene), that reporter gene is expressed and, depending on the level of expression, the promoter strength is inferred.
  • the determination of regulatory properties, in particular promoter properties, essentially comprises three steps. It starts with
  • Step 1
  • Eukaryotic protists are treated with agents / protocols.
  • the mRNA pattern of treated cells is compared with that of the untreated control cells by differential gel electrophoretic analysis (differential display of a Northern blot).
  • differential gel electrophoretic analysis differential display of a Northern blot.
  • nucleic acids whose mRNA signal is either weakened or amplified after "treatment” and which consequently correlate with the defined properties.
  • the following steps serve to identify and characterize the potentially associated 5 ' and 3 ' flanking areas.
  • the cDNA (production, see Maniatis, supra) of the identified mRNA signals is cloned into E. coli-compatible vectors (e.g. pUC18). These clones are sequenced and correlated by means of DNA probes with the corresponding GSP in the Southern blot, as is known to the person skilled in the art (Maniatis, supra).
  • Step 5 DNA sequencing takes place, the 5 ' and 3 ' flanking regions of the identified GSP. As a result, the PCR primers according to the invention can be generated (see step 5). Step 5:
  • the identified 5 ' or 3 ' flanking regions are amplified using a PCR reaction.
  • further PCR primers contain as hybrid primers an overlap to the 5 ' or 3 ' end of the indicator gene used (GFP or GUS in the examples).
  • the telomer primers With the help of the telomer primers, the nucleic acid fragment obtained is amplified in a second PCR step, which has ends with recognition sites for the restriction endonucleases Xbal and Apal and is easily integrated in Smal linearized form into a compatible E. coli vector by means of a DNA ligation reaction can be.
  • This construct is then transformed into E. coli for amplification and obtained in large quantities by isolating the plasmid DNA (pUC 18 or others known to the person skilled in the art for this purpose can be used as an E. coli compatible vector
  • Vectors or plasmids can be used).
  • the expression construct is transformed into protists using the known methods. Analysis of the regulatory strength and properties, preferably
  • Promoter strength and properties are based on the spectroscopic properties of the selected indicator protein.
  • Figure 1 describes the principle of the two-step PCR, which is explained in the following text.
  • SEQ ID No.1 Nucleic acid relating to the 5 ' flanking region (leader region and telomer sequence) consisting of 215 nucleotides.
  • SEQ ID No.2 nucleic acid relating to the 5 ' flanking region (trailer region and telomer sequence) consisting of 261 nucleotides.
  • SEQ ID No.3 "St-gfp": Expression vector consisting of 5 ' and 3 ' flanking region, including nucleic acid coding for GFP, 2-13 Xbal and Apa I, 13-228 leader from alpha I Tubulin S. lemnae + Telomer , 229-946 CDS GFP, 946 - 1204Trailer made of alpha I Tubulin S. lemnae + Telomer, 1205 - 1216 Xbal and Apa I.
  • SEQ ID No.4 "St-gus": Expression vector consisting of 5 ' and 3 ' flanking region, including nucleic acid coding for GUS, 2-13 Xbal and Apa I, 13-228 leader from alpha I Tubulin S. lemnae + Telomer , 229-2037 CDS GUS, 2038 - 2297 Trailer made of alpha I Tubulin S. lemnae + Telomer, 2298 - 2308 Xbal and Apa I.
  • SEQ ID No.5 "St-neo": Expression vector consisting of 5 ' and 3 ' flanking region, including nucleic acid coding for neomycin, 2-13 Xbal and Apa I, 13-228 leader from alpha I Tubulin S. lemnae + Telomer , 229-1023 CDS neomycin gene, 1024 - 1283 trailer from alpha I tubulin S. lemnae + Telomer, 1284 - 1294
  • SEQ ID No.6 Expression vector consisting of 5 ' and 3 ' flanking region, including nucleic acid coding for Zeocin / GFP fusion protein, 2-13 Xbal and Apa I, 13-228 leader from alpha I Tubulin S. lemnae + Telomer, 229- 933 CDS superGFP, 934-1305 CDS Zeocin, 1306 - 1565 Trailer made of alpha I Tubulin S. lemnae + Telomer, 1566 - 1570 Xbal and Apa I.
  • SEQ ID No.7 Expression vector consisting of 5 ' and 3 ' flanking region, including nucleic acid coding for alpha 1 tubulin from S.
  • Expression cassettes are used to transform Stylonychia lemnae, in which the genes for the indicator proteins "GUS” and “GFP” or the genes for the resistance markers neomycin and zeocin are under the control of 5 ' - and
  • the 3 ' located regulatory sequences of the alphal tubulin gene from Stylonychia lemnae are shown in FIG. 3.
  • the expression cassettes, which consist of the resistance marker or indicator gene and the 5 ' and 3 ' located regulatory sequences, were analyzed using a two-step PCR Strategy and then in commercially available pTRACE vectors (Invitrogen) via a
  • step I there were three DNA fragments (alphal tubulin leader, indicator or resistance gene, alpha tubulin
  • step I the DNA fragments are amplified, which are fused in the subsequent PCR reaction (step II).
  • step II the DNA fragments are amplified, which are fused in the subsequent PCR reaction (step II).
  • the three DNA fragments used for the fusion reaction in step II) were obtained in independent PCR reactions in which the DNA templates and primers described below were used under the conditions listed for
  • the first 17 bases stand for the correspondence of the PCR product obtained with the 5 ' end of the GFP ORF.
  • the last 17 bases act as antisense primers to amplify the leader of the alpha tubulin gene.
  • Plasmid pTW54 which was developed by Prof. S. Subramiani (University of California)
  • the first 17 bases are for match with the 3 ' end of the tubulin
  • the first 16 bases are for match with the 5 ' end of the tubulin
  • the first 20 bases are for the overlap with the 3 ' end of the GFP ORF, the last 16 bases are used to amplify the trailer sequence of the tubulin gene
  • step II) the PCR fragments obtained in step I) were fused in a PCR approach and the DNA sequence thus formed by using PCR primers, each of which had the recognition sequence at the ends carry for restriction enzymes, provided at the 5 ' and 3 ' ends with restriction sites.
  • dNTPs 50 ⁇ M in each case After 5 min denaturation, a unit expand high fidelity PCR system was added and subjected to 10 cycles (1 min each 94 ° C., 55 ° C. and 72 ° C.).
  • the constructs were purified by separation in an agarose gel and isolated using a gel extraction kit (Qiagen). Using T4 polynucleotide kinase (Pharmacia), the PCR fragments were then blunt-ended together with the vector pUC18 linearized and dephosphorylated at the Smal interface using the T4 DNA ligase (Pharmacia). This reaction was carried out according to the manufacturer's instructions. The cloning and propagation in E. coli was carried out according to standard protocols (Sambrook et al.). The construction of the rest, used in the description and drawing,
  • PCR primer Forward: 5 ' -C4A4C4A4C4-3 ' (telomeric)
  • the first 17 bases are matched to the 5 ' end of the GFP ORF, the last 17 bases act as a reverse primer to amplify the leader sequence and ORF of tubulin.
  • Plasmid pTW54 provided by Prof. S. Subramani (University of California).
  • PCR primer Forward: 5 ' AAG AAG AAG GTA TGG AAA TGA GTA AAG GAG AAG A-3 '
  • the first 17 bases are for agreement with the 3 ' end of the tubulin
  • the first 16 bases are to match the 5 ' end of the tubulin trailer, the last 20 bases act as a reverse primer to amplify the GFP ORF.
  • Plasmid pTW54 provided by Prof. S. Subramiani (University of California).
  • conjugation two strains of different mating types were put under starvation stress and mixed. The cells were then synchronized by adding a strictly limited amount of nutrients, so that they were synchronized after about five hours. The actual conjugation then started another six hours later. The yield of conjugants was approximately 90%. A further enrichment of the conjugants was possible by filtration through a 70 ⁇ m nylon matrix gauze, which is only permeable to single cells but not to conjugant pairs.
  • the meiotic division of the micronuclei is initiated, the haploid micronuclei are then exchanged between the conjugation partners, and the micronuclei of the partner cells fuse. Then the cells separate again, the newly formed diploid micronuclei share mitotically without simultaneous cell division.
  • One of the daughter cores goes through a series of DNA replications, which eventually lead to the formation of a new macronucleus.
  • the DNA used was obtained either in the course of a PCR reaction with the telomer primer (5 ' -C4A4C4A4C4-3 ' ) (PCR reaction with a primer, since the gene sized pieces from Stylonychia lemnae on both the 5 ' and the Wear 3 ' end telomer sequences !).
  • the PCR products were then applied to the gel
  • Extraction kit (Qiagen) cleaned and used for gene bombardment.
  • the DNA samples were obtained from the plasmids produced according to the described methods by restriction digestion with the restriction endonuclease Apal and subsequent isolation from the gel (see above).
  • the transformed cells Upon completion of macronucleus development (one to two days after bombardment), the transformed cells were exposed to selective conditions. To do this, they were treated with 50 - 100 ⁇ g / ml G418 for 3 to 10 days or for 2 - 4
  • the stably transformed clones were isolated and by detecting the newly introduced foreign information on the DNA (Southern blot, PCR), RNA (Northern blot, primer extension) and protein level (more spectroscopic

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  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Plant Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne des vecteurs d'expression à séquences régulatrices de Stylonychia lemnae pour l'expression de protéine hétérologue dans des protistes eucariotiques, ainsi que leurs optimisations pour la transformation de ces vecteurs, de préférence dans des ciliates, en particulier dans le Stylonychia lemnae. L'invention concerne également un procédé d'identification de séquences régulatrices appropriées des protistes eucariotiques.
PCT/EP1999/007958 1998-10-21 1999-10-20 Vecteurs d'expression renfermant des sequences regulatrices de stylonychia lemnae pour l'expression de proteine heterologue dans des protistes eucariotiques, et procede d'identification de telles sequences regulatrices WO2000023604A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19848486.0 1998-10-21
DE19848485 1998-10-21
DE19848486 1998-10-21
DE19848485.2 1998-10-21

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WO2000023604A1 true WO2000023604A1 (fr) 2000-04-27

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Publication number Priority date Publication date Assignee Title
US7544859B2 (en) 2000-02-09 2009-06-09 Basf Aktiengesellschaft Elongase gene and method for producing multiple-unsaturated fatty acids
CN108823100A (zh) * 2018-08-06 2018-11-16 哈尔滨师范大学 一种浮萍棘尾虫纯培养体系及其制备方法和培养方法
KR20190006802A (ko) 2017-07-11 2019-01-21 현대자동차주식회사 러그를 구비한 자전거 프레임

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US5665565A (en) * 1994-07-12 1997-09-09 The University Of Virginia Patent Foundation Transfection of enteric parasites
DE19626564A1 (de) * 1996-07-03 1998-01-08 Hoechst Ag Genetische Transformation von Ciliatenzellen durch Microcarrier-Bombardement mit DNA beladenen Goldpartikeln

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US5665565A (en) * 1994-07-12 1997-09-09 The University Of Virginia Patent Foundation Transfection of enteric parasites
DE19626564A1 (de) * 1996-07-03 1998-01-08 Hoechst Ag Genetische Transformation von Ciliatenzellen durch Microcarrier-Bombardement mit DNA beladenen Goldpartikeln

Non-Patent Citations (6)

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Title
ASCENZIONI, F. ET AL.: "A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia.", GENE, vol. 46, 1986, pages 123 - 6, XP002128555 *
GAERTIG, J. ET AL.: "High frequency vector-mediated transformation and gene replacement in Tetrahymena.", NUCELIC ACID RESEARCH, vol. 22, 1994, pages 5391 - 8, XP002128557 *
HELFTENBEIN, E. ET AL.: "Nucleotide sequence of a macronuclear DNA molecule coding for alpha-tubulin from the ciliate Stylonychia lemnae. Special codon usage: TAA is not a translation termination codon.", NUCLEIC ACID RESEARCH, vol. 13, 1985, pages 415 - 33, XP002128556 *
MEYERS G ET AL: "TRANSFECTION OF THE HYPOTRICHOUS CILIATE STYLONYCHIA LEMNAE WITH LINEAR DNA VECTORS", GENE,NL,ELSEVIER BIOMEDICAL PRESS. AMSTERDAM, vol. 63, no. 1, pages 31-40, XP002044344, ISSN: 0378-1119 *
SKOVORODKIN, I. ET AL.: "Stable transfection of the hypotrichous ciliate Stylonychia lemnae with Tagged alpha1 tubulin minichromosomes.", EUROPEAN JOURNAL OF PARASITOLOGY, vol. 35, 25 February 1999 (1999-02-25), pages 70 - 80, XP000870238 *
WÜNNING, I.U. ET AL.: "A transformation system for the hypotrychous ciliate Stylonychia mytilus.", EMBO JOURNAL, vol. 2, no. 10, 1983, pages 1753 - 7, XP000870012 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7544859B2 (en) 2000-02-09 2009-06-09 Basf Aktiengesellschaft Elongase gene and method for producing multiple-unsaturated fatty acids
US8933300B2 (en) 2000-02-09 2015-01-13 Basf Aktiengesellschaft Elongase gene, and process for the preparation of polyunsaturated fatty acids
KR20190006802A (ko) 2017-07-11 2019-01-21 현대자동차주식회사 러그를 구비한 자전거 프레임
CN108823100A (zh) * 2018-08-06 2018-11-16 哈尔滨师范大学 一种浮萍棘尾虫纯培养体系及其制备方法和培养方法
CN108823100B (zh) * 2018-08-06 2022-05-17 哈尔滨师范大学 一种浮萍棘尾虫纯培养体系及其制备方法和培养方法

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