WO2000023604A1 - Expression vectors containing regulative sequences of stylonychia lemnae for heterologous protein expression in eukaryotic protists and a method for identifying regulative sequences of this type - Google Patents

Abstract

The invention relates to expression vectors with regulative sequences of Stylonychia lemnae for heterologous protein expression in eukaryotic protists and to the optimisation of the same for transforming these vectors, preferably into ciliates, especially into Stylonychia lemnae. The invention also relates to a method for identifying suitable regulative sequences of eukaryotic protists.

Description

Expression vectors containing regulatory sequences from Stylonychia lemnae for heterologous protein expression in eukaryotic protists and a method for identifying such regulatory sequences

description

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.

A variety of biotechnologically usable microorganisms (e.g. E.coli, S.cerevisiae, P.pastoris, B.subtilis, etc.) are used for so-called 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.

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

Recombination. For the selection of the transformants, the selection via chemical agents, mostly antibiotics, is often chosen. To determine the Expression levels are used so-called indicator genes, ie genes whose gene products can easily be determined qualitatively or quantitatively using spectroscopic methods.

Interestingly, so far it has not been possible to find proteins in eukaryotic

To express protists, especially in ciliates, in sufficient quantities in a functionally heterologous manner.

Yao, M.C. et al., WO97 / 40060 describes ribosomal vectors for the use of antisense strategies in Tetrahymena thermophila. This work is intended to titrate out cellular mRNA from Tetrahymena by hybridization and thus "switch off" genes defined within the framework of an antisense strategy. This shows that in vitro DNA can be transformed into Tetrahymena. The vectors used there are not designed to support the conversion of DNA into proteins, since the necessary control elements (so-called regulative

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 (mediated by the ribosomal origin of replication) can only be modified to a limited extent.

The possibility of introducing DNA into Protozoae in vitro has also been shown by the work of Ascenzioni and Lipps (Gene 46, pp. 123-126, 1986). Plasmids with rDNA sections from Tetrahymena pyriformis were combined with DNA from E. coli, Saccharomyces cerevisiae or Dictyostelium discoideum and introduced into Stylonychia lemnae by means of microinjection. Since indicator proteins have not been used, it is not possible to make any statements about the level of expression of heterologous protein expression. Work on the transformation of Protozoae of the species Stylonychia mytiius was also carried out by Wünning, IU and Lipps, HJ. (EMBO J. 2, 1983), the in vitro DNA transfer also taking place via microinjection. However, data on expression level, optimized control sequences and replication elements are also missing here.

Yu, G.L. and Blackbum, E.H. describe the transformation of Tetrahymena with mutated, circular ribosomal vectors (Mol.Cell.Biol.10, S.2070-2080, 1990). In addition to the disadvantages described above of using vectors which carry rDNA sections for replication support, the problem lies in the fact that efficient selection via resistance markers is not possible. In addition, data on controls of transcription, the use of indicator proteins, the expression levels and the feasibility of heterologous protein expression per se are missing.

Kahn et al. (PNAS 90, 1993) also describe that DNA transformation in Protozoae is possible via microinjection. However, this method of DNA transformation is very inefficient. The authors describe circular vectors that contain rDNA sections from Tetrahymena. Work carried out in parallel with constructs that allow direct incorporation into the genome via homologous

Recombination caused increased transformation rates in comparison. However, the incorporation of foreign DNA into the genome of the target organism runs the risk that the expression of host genes is changed or completely switched off.

Using the electroporation method, Gaertig et al. (NAR 22, 1994) a more efficient transformation method for protists. However, no data on expression level and promoter selection are shown here either. Therefore, no qualitative statement is possible.

US 5,665,565 (Mann, DB, et al.) Describes the expression of foreign proteins in Entamoeba histolytica. During this work, the foreign DNA was flanked by Control elements of a gene from Entamoeba histolytica, introduced into the organism with vectors which contained sections of rDNA. DNA transfer was via electroporation, a method that works more efficiently than microinjection, but is not as successful as gene bombardment. As a human-parasitic, anaerobic protozoa, Entamoeba histolytica, due to its biological characteristics, is not suitable for protein extraction on a larger, industrial scale. In addition, the expression vector based, among other things, on rDNA sequences has the disadvantages of ribosomal expression vectors already described above. The extent to which the work can be transferred to other Protozoae is also unclear.

Patent application DE19626564 describes the basic feasibility of heterologous protein expression in Stylonychia lemnae using the method of gene bombardment to transform protists.

Haddad, A. and Turkewitz, A.P. describe experiments in which a ribosomal plasmid is used in order to use GFP to investigate the expression characteristics of the regulatory sequence of the GRL1 gene locus in Tetrahymena thermophila (PNAS 94, 1997). This work uses neither optimized control sequences for gene expression nor optimized ones

Replication sequences (ribosomal replication elements only).

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. Mostly, 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

Properties that 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.

In the somatic macronucleus genomes of most eukaryotic protists, all genes are more or less amplified. This leads among other things to a high expression rate of these genes even under normal conditions. In some species, up to 98% of the micronucleus genome is eliminated, intervening sequences are cut out of genes and coding regions can be rearranged completely (so-called "gene scrambling"; see D.C. Hoffmann; D.M.

Prescott (1997), NAR 25 (10), 1883).

However, the use of eukaryotic protists as systems for the production of recombinant proteins has so far encountered a number of difficulties.

On the one hand, the methods known in the prior art, which are available for the transformation of eukaryotes, lead to sufficiently high, transformation rates in eukaryotic protists only in a few exceptional cases. This is especially true for ciliates and especially for Stylonychia lemnae.

In the prior art, an insufficient heterologous expression level in the host organism is obtained for eukaryotic protists. In addition, there have so far been hardly any suitable selection markers for transformation attempts with protists, since some selection systems which are generally used successfully for eukaryote transformations cannot be used with protists. In addition, the eukaryotic protists have a codon usage (also codon preference) that differs significantly from that of the other eukaryotes.

The occurrence of "inclusion bodies" is also known, 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.

It has now surprisingly been found that the heterologous protein expression in the presence of the expression vector according to the invention in eukaryotic

Protists succeed while eliminating the disadvantages mentioned.

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.

The object is achieved by 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

Identification of suitable regulatory sequences.

“Protists” in the sense of this invention are eukaryotic protists. In the broadest sense, 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. In the narrower sense, 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.

Within the realm of the Protists, especially Protozoa, details of the classification are still discussed and systematized (Honigberg et al. (1964): A revised ciassification of the phylum Protozoa; J. Protozool. 11; 7; Levine et al. (1980 ): A newly revised ciassification of the Protozoa; J. Protozool., 27; 37). The common classification of eukaryotic protists is according to Cavalier-Smith (Cavalier-Smith, T., 1995; Cell cycles, diplokaryosis and the archezoan origin of sex; Arch. Protistenk., 145 (3-4), 189-207) into 6 kingdoms and 33 phyla:

Kingdom Archezoa:

Archamoebae, Metamonada, Microsporidia

Kingdom Protozoa:

Percolozoa, Parabasalia, Euglenozoa, Mycetozoa, Entamoebia, Opalozoa, Dinozoa, Apicomplexa, Ciliophora, Haplosporidia, Paramyxia, Rhizopoda, Reticulosa, Heliozoa, Radiozoa, Amoebozoa, Choanozoa

Kingdom Chromista:

Cryptista, Heterokonta, Haptophyta Kingdom Fungi:

Chytridiomycota, Zygomycota, Ascomycota, Basidiomycota

Kingdom Animalia: Myxozoa, Mesozoa

Kingdom Plantae:

Chlorophyta, Charophyta, Glaucophyta, Rhodophyta

Ciliates (synonym Ciliophora, Ciliata), which have a so-called core dimorphism, can be seen as a particularly advantageous host cell. This means that the DNA is present in two differentiated cell nuclei. In addition to the small, transcriptionally inactive, mostly diploid micronucleus, there is the DNA-rich, transcriptionally active macronucleus. The micronucleus mainly has generative functions. In the course of conjugation, 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.

For the purposes of this invention, 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.

For the purposes of this invention, 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

For the purposes of this invention, 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

"Gene-sized-pieces (GSP)" in the genomic DNA.

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.

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.

In the sense of a transcription fusion, the expression vector according to the invention allows the expression of a protein encoded by the foreign gene as an authentic, biologically active enzyme. For this purpose, 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. In particular, 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.

In a special embodiment, 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.

In ciliates, especially Stylonychia lemnae, the genomic DNA is in the form of "gene-sized-pieces (GSP)". These are independent units capable of replication which, in addition to the 5 ^' and 3 ^' flanking regions mentioned, have terminal specific telomer sequences. The use of these telomer sequences according to the invention enables a further advantageous increase in expression in eukaryotic protists. The expression vector according to the invention represents such a GSP unit.

In the direction of transcription (5 ^' to 3 ^' ) before the region / sequence of a eukaryotic gene coding for a protein, which begins with the ATG start codon, is the 5 ^' flanking region of the gene which is essential to the invention and which is not transcribed. This is the promoter / enhancer region according to the invention in the context of this invention. Within this 5 - untranslated region ^'flanking region of the so-called leader or ^5' lies (synonym 5 ^'- untranslated region, ^"5' UTR"). The leader area, which with the ATG

Start codon ends, plays a role in mRNA translation with effect for the cap structure. In the direction of transcription (5 ^' to 3 ^' ) behind the region / sequence coding for a protein of a eukaryotic gene, 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.

In the context of this invention, the native 3 'and ^5' are - flanking regions from the alpha tubulin gene of Stylonychia lemnae after

Methods known to those skilled in the art, but the selected sequences SEQ ID No. are particularly preferred. 1 and SEQ ID No. Second

Due to the redundant information of the codons (base triplets) which code for an amino acid, 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 ").

The 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.

"Native" in the sense of the present invention is referred to as 3'-non-coding and

5 'non-coding nucleic acid regions which originate from the host organism, a eukaryotic protist, preferably a ciliate, particularly preferably Stylonychia lemnae.

To carry out the invention, 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). For this purpose, according to 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. When using the 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

Expression of the 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.

In a particularly advantageous embodiment of the invention, the co-transformation is carried out instead of the (co) vector pST-neo with the (co) vector pSt-tracer (SEQ ID No. 6). In this vector, 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. In the case of co-transformation, 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.

The basic applicability of a transformation method for protists in the context of microcarrier bombardment with DNA-loaded gold particles is described in US Pat. No. 5,036,006 or WO 98/01572 and can be carried out accordingly. It is also conceivable to introduce the expression vector according to the invention by means of electroporation known to the person skilled in the art.

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.

In a further preferred embodiment, 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. After expression of the designated nucleic acid, a fusion protein is obtained from the desired protein and an N-terminally fused peptide which contains the histidines mentioned. As a result, 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 can also be easily purified using affinity chromatography and detected using a colorimetric test or an immunoassay.

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. 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.

In a particular embodiment, eukaryotic protist cells, preferably ciliates, particularly preferably Stylonychia lemnae, can be transformed which are at the time of conjugation with the corresponding conjugation partner in the phase of plant formation or macronucleus maturation and can thus be transformed particularly efficiently.

In this context, it is essential that the invention

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

Use of an expression vector according to the invention for the production of a desired protein. In this respect, 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. in the

Following this process - known as DNA transformation - the organisms that have taken up the expression construct must be selected. To do this, a number of so-called resistance markers are used, which enable growth in the presence of selective agents - mostly antibiotics or related substances. Important prerequisite for direct

Selection via resistance markers is the functional expression of the underlying resistance principle. Surprisingly, it was shown in the present work that the selection markers Zeocin and Neomycin, which were known from applications in certain mammalian cell lines, bacteria or yeasts, successfully isolated under suitable reaction conditions for isolating transformants in

Have Stylonychia lemnae inserted.

Following the selection of the transformants, the expression level of the desired protein is determined. In heterologous protein expression, the focus is on a high protein production rate. An often decisive factor here is the selection of a suitable promoter. In addition to a high level of mRNA of the target protein, 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

Sequences in eukaryotic protists, preferably S. lemnae, with the aim of integrating promoter-strong regulative sequences into the expression vector according to the invention, in addition to the sequences SEQ ID No provided. 1 and SEQ ID No. Second

Many genes are regulated almost exclusively at the level of transcription. This means that the decision about whether and in what concentration Protein occurs in the cell at a defined point in time, in many cases it can be determined almost exclusively by regulatory sequences, in particular by means of promoters.

The focus is on the yield of functional hetroiogenic protein product, which is determined decisively by the promoter used. In addition to the relevant promoter strength, the growth phase in which the desired foreign protein is produced is often of interest. This requires so-called inducible promoters. These are regulatory sequences that, under certain conditions (addition of a so-called

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.

Another frequently used promoter is the so-called

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.

With the help of the well-known recombination technology, 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).

In this respect, 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.

Eukaryotic protists, in particular, which have "Gene Sized Pieces (GSP)" are particularly suitable as promoter test systems for regulatory sequences.

The invention therefore also relates to a promoter test system with the method steps:

(a) Identification of nucleic acid with defined properties in eukaryotic protists;

(b) sequencing the 5 ^' and 3 ^' flanking regions;

(c) PCR amplification of the regions mentioned in (b) using telomeric primers and hybrid primers (indicator gene / 5 ^' and 3 ^' flanking region) and in the second step of the nucleic acid fragment obtained (cf.

Comments on the 2 step PCR);

(d) E. coli compatible test vector containing the nucleic acid fragment mentioned in (c);

(e) Transformation into eukaryotic protists and determination of the regulatory strength, preferably promoter strength.

For the purposes of this subject matter of the invention, 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 means that the promoter strength has an immediate effect on nucleic acid lying in the 3 ^' direction behind the promoter. 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. Regarding 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.

For the purposes of this invention, nucleic acids with regulatory activity from eukaryotic protists are identified and isolated, which are characterized by defined properties.

Such defined properties are, for example, without concluding character:

Resistance to heat, cold, pests and other functional motives. 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

Cloning of the regulatory DNA section to be tested into the relevant promoter test vectors and the amplification of these constructs in E. coli. After transfer of this DNA into the desired host organism (DNA transformation), the transformants are selected. The level of expression of the indicator proteins is then determined qualitatively and quantitatively, in particular using spectroscopic methods. In summary, the following steps are carried out:

Step 1 :

Eukaryotic protists are treated with agents / protocols. To

Standard protocols (see Molecular Cloning, A laboratory Manual, Second Edition,

J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press,

1989, Cold Spring Harbor, New York, USA) the mRNA as well as the genomic

DNA of the treated and untreated (control) eukaryotic protists was obtained.

Step 2:

To identify putative regulatory sequences, 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). Of interest are the 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.

Step 3:

First, 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 4:

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. In addition to the terminal PCR telomer primers, 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). 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.

Step 6:

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).

Step 7:

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.

The following figures and examples are intended to explain the invention in more detail without restricting it thereto. Description of the figures and the most important sequences:

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

Xbal and Apa I.

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. lemnae and GFP, 2-13 Xbal and Apa I, 13-228 leader from alpha I tubulin S. lemnae + Telomer, 229- 1563 CDS alpha 1 tubulin gene from S. lemnae, 1564-2280 CDS GFP, 2281 - 2540 Trailer from alpha I Tubulin S. lemnae + Telomer, 2541 - 2551 Xbal and Apa I.

Examples:

Example 1: Construction of the expression vectors used according to the invention (vector,

Plasmid):

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

DNA ligation reaction built in.

Two-step PCR strategy

In the two-step PCR strategy, the DNA fragments for the resistance or indicator gene and the DNA sections from the

5 ^' - and 3 ^' - located regulatory sequences of the alphal tubulin gene amplified by PCR. The fragments were each constructed in such a way that a 16-20 bp overhang was inserted which corresponded to the corresponding sequence in the neighboring fragment (see FIG. 1). At the end of step I) there were three DNA fragments (alphal tubulin leader, indicator or resistance gene, alpha tubulin

Trailer). An overlapping region of 34-40 bp (16-20 bp each from the leader or trailer and 16-20 bp from the central resistance or In step II), indicator gene) then ensured, in a subsequent PCR reaction, by using PCR primers with terminal restriction sites matched to the vector, that an overall expression cassette was obtained which could be easily incorporated into the corresponding vector (see also FIG 1 ).

The details of the two-step PCR strategy for the extraction of the expression cassette "ST-gfp" are described below, in which the GFP ORF is under the control of the 5 ^' and 3 ^' located control sequences of the alphal tubulin gene from Stylonychia lemnae .

Step I):

As described above, in step I) the DNA fragments are amplified, which are fused in the subsequent PCR reaction (step II). By using long PCR primers that carry a 16-21 bp overhang to the others

Fits fragments that are fused in step II), the fusion reaction is out

Step II) only possible.

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

PCR reactions were used.

1. Construction of St-gfp

1.1 Amplification of the leader sequences of the alphal tubulin gene from Stylonychia lemnae with overlap to the 5 ^' end of the ORF of GFP (S65T):

Template:

Gen-sized piece of the alphal tubule gene from Stylonychia lemnae, which is cloned into the pBluescript vector (Helftenbein and Müller, 1988)

PCR primer:

Forward: 5 ^' -C4A4C4A4C4-3 ^' (telomeric) Reverse: 5 ^' -TCT TCT CCT TTA CTC ATG ATG AAG AGT TGA TTT G-3 ^'

(tugfprl)

With the primer tugfprl, 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.

1.2 Amplification of the GFP ORF (S65T) with a match to the ends of the leader or trailer sequences of the alpha tubulin gene from Stylonychia lemnae.

Template:

Plasmid pTW54, which was developed by Prof. S. Subramiani (University of California)

Was made available.

PCR primer:

Forward: 5 ^' -CAA ATC AAC TCT TCA TCA TGA GTA AAG GAG AAG A-3 ^'

(tugfpfl)

The first 17 bases are for match with the 3 ^' end of the tubulin

Leaders, the last 17 bases serve to amplify the GFP ORF (S65T) as a forward primer.

Reverse: 5 ^' -GTG GGC GAA TGT ATG CTC ATT TGT ATA GTT CAT CCA -3 ^'

(gfptur2)

The first 16 bases are for match with the 5 ^' end of the tubulin

Trailers, the last 20 bases serve as reverse primers to amplify the GFP ORF (S65T).

1.3 Amplification of the trailer sequences of the alpha tubulin gene from Stylonychia lemnae in agreement with the 3 ^' end of the GFP ORF

Template:

Gene sized piece of the alpha tubulin gene from Stylonychia lemnae, cloned into the pBluescript vector (Heiftebein and Müller, 1988) PCR primer:

Forward: 5 ^' -TGG ATG AAC TAT ACA AAT GAG CAT ACA TTC GCC CAC-3 ^' (gfptuß)

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

Reverse: 5 ^' -C4A4C4A4C4-3 ^' (Telomer primer)

PCR conditions for the reactions from step I):

A 50 μl mixture contained (in the Expand High Fidelity PCR buffer system):

Template DNA: 50ng

PCR primer: 20 pmol dNTPs in each case: 50 μM each

Expand High Fidelity PCR System: 0.5 units

Temperature profile on the thermal cycler:

Denaturation: 1 min at 94 ° C

Annealing: 1 min at 56 ° C DNA synthesis: 1 min at 72 ° C

Number of reaction cycles: 35

Synthesis time in the last cycle: 15 min at 72 ° C

The PCR products obtained according to the approaches described above were purified in the gel (Gel Extraction Kit, Qiagen) and according to the descriptions in step

II) further processed.

Step II):

As explained above, in 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.

For a reaction in a 50 μl mixture, Expand High Fidelity PCR buffers were used:

Template:

50 ng of an equimolar mixture of the three PCR obtained in step I)

Products. dNTPs, 50 μM 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.).

Subsequently, 20 pmol of the telomeric primer (XbaApatel) was added

Restriction interfaces for Xbal and Apal in the 5 ^' region (sequence: 5 ^' -GTC TAG

AGG GCC CCC CAA AAC CCC AAA ACC-3 ^' ) added and subjected to a PCR with the following settings:

Denaturation: 1 min at 94 ° C

Annealing: 1 min at 55 ° C

DNA synthesis: 2 min at 68 ° C number of reaction cycles: 30

Synthesis time in the last cycle: 15 min at 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,

Expression constructs were made according to the procedure described above. The primers used in each case are shown below. 2. Construction of "St-tuegfp"

2.1 Amplification of the leader sequences and the ORF of the alphal tubule gene from Stylonychia lemnae which correspond to the 5 ^' -GFP (S65T) ORF.

Template:

Gene sized piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988 (supra))

PCR primer: Forward: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

Reverse: 5 ^' -TCT TCT CCT TTA CTC ATT TCC ATA CCT TCT TCT T-3 ^'

(tuegfpr3)

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.

2.2 Amplification of the GFP ORF in agreement with the 3 ^' ORF of the tubulin gene from Stylonychia lemnae

Template:

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 ^'

(tuegfpf3)

The first 17 bases are for agreement with the 3 ^' end of the tubulin

ORF, the last 17 bases serve as a forward primer to amplify the GFP ORF. Reverse: 5 ^' -GTG GGC GAA TGT ATG CTC ATT TGT ATA GTT CAT CCA-3 ^'

(gfptur2) 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.

2.3 Amplification of the trailer sequences of the alpha tubulin gene from Stylonychia lemnae with overlaps to the 3 ^' end of the GFP ORF

Template:

Gene sized piece of alpha tubuingen from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988 (supra))

PCR primer:

Forward: 5 ^' -TGG ATG AAC TAT ACA AAT GAG CAT ACA TTC GCC CAC-3 ^' (gfptuß) The first 20 bases serve to overlap with the 3 ^' end of the GFP ORF, the last 16 bases act as forward primers, to amplify the tubule gene trailer sequence. Reverse: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

3. Construction of St-gus:

3.1 Amplification of the leader sequence of the alphal tubulin gene from Stylonychia lemnae with overlap to the 5 ^' end of the GUS ORF

Template: Gene Sized Piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988)

PCR primer:

Forward: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

Reverse: 5 ^' -GGG GTT TCT ACA GGA CGT AAC ATG ATG AAG AGT TGA TTT G-3 ^' (tugudr1) 3.2 Amplification of the GUS ORF with overlap to the leader and trailer sequences of the alphal tubule gene from Stylonychia lemnae

Template: pRT103gus (courtesy of Jefferson)

PCR primer:

Forward: 5 ^' -CAA ATC AAC TCT TCA TCA TGT TAC GTC CTG TAG AAA CCC C-3 ^' (tugusf1) Reverse: 5 ^' -GTG TGG GCG AAT GTA TGC TCA TTG TTT GCC TCC CTG

CTG-3 ^' (gustur2)

3.3 Amplification of the trailer sequences of the alphal tubule gene from Stylonychia lemnae with overlap to the 3 ^' end of the GUS ORF

Template:

Gene sized piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988)

PCR primer:

Forward: 5 ^' -CAG CAG GGA GGC AAA CAA TGA GCA TAC ATT CGC CCA

CAC-3 ^' (gustuf2)

Reverse: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

4. Construction of St-tracer

4.1 Amplification of the leader sequences of the alphal tubule gene from Stylonychia lemnae with overlap to the 5 ^' -GFPsuper ORF

Template: Gene Sized Piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988) PCR primer:

Forward: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

Reverse: 5 ^' -CTT CTC CTT TGC TAG CCA TGA TGA AGA GTT GAT TTG-3 ^'

(tutracerl)

4.2 Amplification of the GFP / Zeocin ORF with overlap to the leader and trailer sequence of the alphal tubule gene from Stylonychia lemnae

Template: pCMV tracer (Invitrogen)

PCR primer:

Forward: 5 ^' -CAA ATC AAC TCT TCA TCA TGG CTA GCA AAG GAG AAG-3 ^' (tutracef 1) Reverse: 5 ^' -GTG GGC GAA TGT ATG CTC AGT CCT GCT CCT CGG CCA-3 ^'

(tracetur2)

4.3 Amplification of the trailer sequence of the alphal tubulin gene from Stylonychia lemnae with overlap to the 3 ^' end of the Zeocin ORF

Template:

Gene sized piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988)

PCR primer:

Forward: 5 ^' -TGG CCG AGG AGC AGG ACT GAG CAT ACA TTC GCC CAC-3 ^'

(tracetuf2)

Reverse: 5 ^' -C4A4C4A4C4-3 ^' (telomeres)

5. Construction of St-neo

5.1 Amplification of the leader sequences of the alphal tubule gene from Stylonychia lemnae with overlap to the 5 ^' region of the Neo ORF Template:

Gene sized piece of the alpha tubule gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988)

PCR primer:

Forward: 5 ^' -C4A4C4A4C4-3 ^' (telomeric)

Reverse: 5 ^' ATC CAT CTT GTT CAA TCA TGA TGA AGA GTT GAT TTG-3 ^'

(tuneorl)

5.2 Amplification of the Neo ORF with overlap to the leader and

Trailer sequences of the alphal tubule gene from Stylonychia lemnae

Template:

Plasmid pTW54, provided by Prof. S. Subramiani (University of California).

PCR primer:

Forward: 5 ^' -CAA ATC AAC TCT TCA TCA TGA TTG AAC AAG ATG GAT-3 ^' (tuneofl) Reverse: 5 ^' -GTG GGC GAA TGT ATG CTC AGA AGA ACT CGT CAA GAA-3 ^'

(neotur2)

5.3 Amplification of the trailer sequences of the alphal tubule gene from Stylonychia lemnae with overlap to the 3 ^' end of the Neo ORF

Template:

Gene-sized piece of the alpha tubulin gene from Stylonychia lemnae, cloned into the pBluescript vector (Helftenbein and Müller, 1988) PCR primer: Forward: 5 ^' -TTC TTG ACG AGT TCT TCT GAG CAT ACA TTC GCC CAC-3 ^'

(neotuf2) Reverse: 5 ^' -C4A4C4A4C4-3 ^' (telomere) 6. Conjugation of Stylonychia lemnae

For 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 μm nylon matrix gauze, which is only permeable to single cells but not to conjugant pairs.

When the cells are paired, 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 complete macronucleus development takes approx. 100 hours (calculated from the time of the exconjugant separation). In this process, i.a. Genes - like nonsense DNA - were cut out of the micronuclear chromosome, internal DNA sequences were removed by splicing steps, and genes were amplified and telomeric repeats attached.

The efficiency of the transformation is closely linked to the stage of

Macronucleus ripening. Particularly high transformation rates (higher than 65%) are achieved if the transformation is carried out after the appearance of the polytanic chromosomes (approx. 50 to 60 hours after separation of the exconjugants). 7. Gene bombardment transformation

The transformation of Stylonychia lemnae with the DNA fragments or vectors used in the present patent specification was carried out in accordance with patent specification WO

98/01572.

DNA samples:

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. Alternatively, 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).

Amounts of DNA:

For a transformation approach, 10 μg DNA were loaded onto 3 mg gold particles (1-1.6 μm). This amount was enough for four to five bombardment experiments. In the case of co-transformation, 5 μg of the two constructs were used in each case.

Selection of transformants using a selective agent (Zeocin or G418)

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 μg / ml G418 for 3 to 10 days or for 2 - 4

Treated for weeks with 500 μg / ml Zeocin until the successfully transformed cells were completely selected.

Following the selection, 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

Evidence, e.g. verified by fluorescence microscopy, fluorimetric measurements).

Claims

claims 1. Expression vector for eukaryotic protists containing a nucleic acid coding for a protein, characterized in that native flanking regulatory sequences selected from the 3 ^' and 5 ^' ends of this nucleic acid Stylonichia lemnae with terminal telomeric sequences are included. 2. Expression vector according to claim 1, characterized in that said flanking regulatory sequences from the alpha tubule gene of Stylonychia lemnae is available, selected from the nucleic acids SEQ ID. No 1 and SEQ ID No. 2, with SEQ ID No. 1 and SEQ ID No. 2 is part of the claim. 3. Expression vector according to claim 1-2, characterized in that said flanking regulatory sequences at least about 50, preferably Contain 150 to approx. 500, particularly preferably 200 to 300 nucleotides, selected from regulatory sequences of the alpha-tubulin gene from Stylonychia lemnae. 4. Telomeric sequences according to claim 1 to 3, characterized in that the telomeric sequence is 5 ^' C4A4C4A4C4 and 3 ^' G4T4G4T4G4 5. Expression vector according to claim 1 to 4, characterized in that the eukaryotic protist originates from the Protozoa realm. 6. Expression vector according to claim 1 to 5, characterized in that the eukaryotic protist is a ciliate. 7. Expression vector according to claim 1 to 6, characterized in that the eukaryotic protist is Stylonychia lemnae. 8. Expression vector according to one of claims 1-7, characterized in that the nucleic acid codes for a human, animal, vegetable or bacterial protein. 9. Expression vector according to claims 1-8 containing as a resistance gene and / or Indicator gene is a DNA segment which codes for a fusion protein from said nucleic acid according to one of Claims 1 to 4 and / or Zeocin and / or Neomycin and / or GFP and / or GUS. 10. Expression vector according to one of claims 1-9, characterized in that an additional functional nucleic acid is contained, which for an oligopeptide from at least 4 histidines, preferably from about 6 histidines and / or for the glutathione-S-transferase and optionally encodes a protease interface. 11. Eukaryonic protist obtainable by transformation by means of an expression vector according to claims 1-10. 12. A method for producing eukaryotic protists, characterized in that a vector according to any one of claims 1-1 1 together with a covector containing a nucleic acid according to SEQ ID No. 5 and SEQ ID No. 6, where SEQ ID No. 5 and SEQ ID No. 6 Part of the claim is introduced or transformed or conjugated into eukaryotic protists, preferably ciliates, in particular Stylonychia lemnae, and the expressed proteins are isolated. 13. A process for the production of eukaryotic protists, characterized in that the eukaryotic protist cells are in the phase of plant formation or macronucleus maturation by previous conjugation with the corresponding conjugation partner and can thus be transformed particularly efficiently. 14. Use of an expression vector according to any one of claims 1-13 for the production of a protein. 15. Use of an expression vector according to any one of claims 1- 14 for the functional heterologous production of a protein. 16. Use of an expression vector according to any one of claims 1-15 for overexpression, wherein 7-40%, preferably 7-15% of the expressed foreign protein is present in the host cell. 17. A method for identifying a regulatory sequence according to claims 1 to 4, comprising the steps: (a) identification of nucleic acid with defined properties; (b) sequencing the 5 ^' and 3 ^' flanking regions; (c) PCR amplification of the regions mentioned in (a) using telomeric Primer and hybrid primer (indicator gene / 5 ^' - and 3 ^' - flanking Area) and in the second step of the nucleic acid obtained Fragments; (d) E. coli compatible test vector which contains the nucleic acid Contains fragment; (e) Transformation into eukaryotic protists and determination of the regulatory strength.