Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungal isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
  • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
  • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/90—Isomerases (5.)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi

Definitions

• the invention relates to a DNA sequence which comprises the triose phosphate isomerase gene including its regulatory sequences, a DNA sequence which is active as a promoter, expression and secretion systems containing this sequence, host cells transformed with this DNA, the use of the sequences for in Yeast cells active expression and secretion systems and methods for the production of polypeptides and RNA using such systems.
• yeasts are also considered, in particular the widespread yeast Saccharomyces cerevisiae, the genome of which is now known and for which some vectors and expression systems are available.
• K.marxianus can use a variety of carbon and energy sources for growth and is not very temperature sensitive.
• Kluyveromyces marxianus can grow at temperatures up to 45 ° C and therefore offers advantages in this respect compared to the more temperature-sensitive Saccharomyces strains in breeding.
• the cells of fast growing K.marxianus btammen can divide every b minutes under optimal conditions.
• the object of the invention was therefore to provide promoters with improved effectiveness which can control the expression of proteins and peptides in yeast cells. Another task was to provide vectors with which polypeptides and proteins can be expressed and the expressed products can be removed from the cell.
• the invention therefore relates to the provision of a new DNA sequence, new regulatory elements, a method for removing proteins from the cell and the provision of suitable expression cassettes, plasmids and microorganisms.
• a DNA with a sequence according to SEQ ID No. 1 or a partial sequence thereof, preferably with at least 10, more preferably at least 100 nucleotides is provided:
• the DNA sequence according to SEQ ID No. 1 is a nucleic acid sequence that encompasses the regulatory regions and the open reading frame for the enzyme triosephosphate lawn coded. In the range of nucleotides 1 to 1 1 1 2 there are active regulatory regions of this gene as a promoter.
• triose phosphate isomerase (hereinafter also referred to as TPI) is a glycolytic enzyme that is involved in the breakdown of glucose and fructose, which takes place in cells for energy, and is therefore widespread.
• the triose phosphate isomerase also serves to isomerize the dihydroxyacetone phosphate formed in the cleavage of fructose-1,6-biphosphate in addition to the glyceraldehyde-3-phosphate to glyceraldehyde-3-phosphate, which is then converted into pyruvate over several stages with energy generation.
• the enzyme is also involved in the metabolism of complex lipids. It is a homodimer, the subunits of which consist of approximately 250 amino acids.
• triosephosphate isomerase (EC 5.3.1 .1) from the yeast type Kluyveromyces marxianus var. Marxianus was elucidated by the inventors and the latter is given in SEQ ID No. 1. Sequences of the TPI enzyme in other organisms are known; the sequence for TPI in S.cerevisiae has been elucidated and given the number YDR050CCDS. In a sequence comparison, it was found that the agreement of the DNA sequences encoding TPI in different microorganisms is high with regard to the open reading frame, but low with regard to the regulatory sequences.
• a further aspect of the invention therefore relates to the provision of a new promoter which has the nucleotides 1 to 1 1 1 2 of SEQ ID No. 1 or parts thereof which are active as a promoter.
• the promoter according to the invention can be used in a manner known per se in a functional connection with foreign genes.
• a DNA sequence with nucleotides 1 to 1 1 1 2 of SEQ ID No. 1 is also referred to below as a promoter sequence.
• a promoter which is active in yeasts and which provides an expression system which is very variable. It is suitable for Kluyveromyces and other types of yeast, among others, and can e.g. for yeast types such as Saccharomyces cerevisiae and Kluyveromyces lactis. Experiments, which are described in the experimental part, have shown that under the control of the promoter according to the invention, a highly effective expression of foreign protein takes place. , ⁇ i
• the new constitutive promoter provided according to the invention is suitable for the expression of recombinant proteins in yeast cells.
• a promoter is understood here to mean a DNA sequence from which the transcription of a gene is controlled.
• the term “part active as promoter” means a partial sequence of the promoter which, in conjunction with an open reading frame, leads to expression of the polypeptide encoded by the reading frame.
• the promoter which regulates the expression of TPI in K.marxianus also referred to below as the KmTPI promoter, has seven possible transcription start sites. These are designated in the sequence from SEQ ID No. 1 as transcription start # 1 to # 7. It was also found that different microorganisms use different sequence motifs as the starting point for transcription for expression. The performance of the promoter depends, among other things, on which of the transcription start points are available for the expression, and therefore the strength of the promoter can be adjusted by selecting the sequence sections for the microorganism in which the expression is to take place.
• the KmTPI promoter in Kluyveromyces marxianus acts so strongly that a portion of the promoter DNA with only one transcription start site leads to the expression of a polypeptide.
• a sequence is preferably used as the promoter which comprises at least five transcription start sites, for example a sequence with at least nucleotides 352 to 1112 of SEQ ID No. 1.
• the KmTPI promoter provided according to the invention is also functional in Saccharomyces. In order to achieve satisfactory expression, however, it is recommended to use the KmTPI maximal promoter with nucleotides 1 to 1 1 1 2 of SEQ ID No. 1 in organisms of this genus.
• nucleic acid sequences with promoter activity include those sequences which have arisen by modification, substitution, deletion or insertion or combinations thereof.
• the promoter sequence can also be provided with further regulatory upstream sequences with enhancer, activator and / or repressor functions.
• Sequences with promoter activity are those sequences which comprise part of the claimed sequence or a derivative thereof which still acts as a promoter for the expression of proteins
• sequences are also considered which are associated with the claimed nucleotide sequence or the parts thereof, e.g. of the promoter sequence have a homology of at least 70%, more preferably at least 90% and in particular at least 95%, as long as the respective sequences also have a comparable biological function.
• the homology is determined in the usual way using the usual algorithms.
• the invention also includes those sequences which hybridize to the sequences according to the invention under stringent conditions.
• those nucleic acids which encode polypeptides homologous to TPI are also considered, in particular those with at least 80% homology.
• the term "homologous polypeptide” includes herein Description A polypeptide that has essentially the same amino acid sequence and essentially the same biological activity as the claimed polypeptide. A homolog can differ from the starting polypeptide in that it has more, fewer or different amino acids, but the function is retained. Those skilled in the art are familiar with methods for producing appropriately modified polypeptides.
• the promoter according to the invention is provided in a manner known per se, either by isolating the naturally occurring sequence, which is preferred, or by producing the sequence by genetic engineering or synthesizing it chemically. Methods for obtaining or synthesizing are known to the person skilled in the art and do not require any further explanation here. It is only essential that the promoter of the triose phosphate isomerase from K. marxianus, with the nucleotides up to and including 1 1 1 2 from SEQ ID No. 1, or an active part thereof is used.
• an expression system or an expression cassette which contains the promoter sequence as defined above or a part thereof which is active as a promoter, a terminator and optionally further regulatory sequences such as cer, and has an insertion cloning site.
• the desired gene to be expressed or the DNA for a foreign protein to be expressed can be inserted into the insertion cloning site and can be transcribed under the control of the promoter according to the invention.
• the expression cassette according to the invention comprises a promoter sequence or a part thereof which is active as a promoter, an insertion cloning site into which the polynucleotide for the protein to be expressed can be cloned, and a nucleotide sequence which acts as a terminator.
• Sequences suitable as an insertion cloning site are known to the person skilled in the art and do not require any further explanation here.
• the sequence which acts as a terminator when expressing TPI can be used as the terminator, for example.
• the latter comprises nucleotides 1860 to 21 63 of SEQ ID No. 1 or parts thereof active as terminator. Good results have also been achieved when using the terminator region of the endopolygalacturonase gene from Kluyveromyces marxianus.
• the expression cassette according to the invention can be used in a variety of ways.
• the insertion cloning site is an interface where the sequence can be cut open and the polynucleotide for the desired protein or peptide can be ligated in.
• the protein is produced intracellularly during expression and is not removed from the cell. After disruption of the cell, it can then be obtained in a manner known per se.
• This embodiment is suitable both for small peptides and proteins that are unstable outside the cell and for proteins that are generally located intracellularly.
• the expression cassettes according to the invention can be used for the expression of DNA sequences in cells, in particular yeast cells.
• the expression cassettes according to the invention are particularly suitable for expression in yeast cells of the genera Saccharomyces and Kluyveromyces, e.g. S. cerevisieae, K. lactis, K. marxianus and others.
• the expression cassette according to the invention is suitable both for incorporation into autonomously replicating plasmids and for incorporation into yeast chromosomes via integrative vectors.
• an expression cassette into which the desired polynucleotide for expressing a peptide or protein has been ligated in an E. coli plasmid and then to obtain the E. coli plasmids, the expression cassette with suitable restriction endonucleases for which Interfaces at the edges of the expression cassette are provided to be cut out and the expression cassette inserted into a yeast vector.
• the vectors usually contain selection markers in order to be able to select successfully transformed cells in a manner known per se.
• the plasmids can optionally be propagated in E. coli and then used in Kluyveromyces marxianus or another K / uyveromyces stavn or also another yeast strain.
• known plasmids based on the Kluyveromyces drosophi / arum-P ⁇ asm ⁇ of pKD1 can be used as the transformation system.
• Descendants of this plasmid are suitable for use in Kluyveromyces marxianus and, when using the expression system according to the invention, lead to an effective expression of foreign proteins in the corresponding host.
• the expression cassette including the polynucleotide to be expressed, can be cut out of the plasmids according to the invention, prepared above, and brought into direct contact with yeast cells as linear or circularized DNA as an integration cassette in order to be taken up by them. Stable incorporation into the host cell can then take place via homologous recombination, provided that some of the nucleic acids are homologous to the host cell. Due to homology of parts of the expression cassette, e.g. of the TPI gene or the regulatory sequences thereof, with the genome of the yeast, the DNA is then taken up in a part of the treated cells into the corresponding chromosomes by exchange with the corresponding sequences.
• the expression cassette according to the invention is stably built into chromosomes and, if the cells are grown under optimal conditions, leads to a good yield of the desired protein.
• the number of copies of the system can be adjusted depending on the type of protein or peptide to be expressed. If a higher number of copies is desired, sequences of a gene which is present in a larger number of copies in the chromosome set, e.g. for rDNA, ligated to cause a higher number of exchange events.
• a marker gene can also be incorporated into the sequence so that the successfully transformed cells can be selected. NEN. Methods and markers suitable for this are known to the person skilled in the art and do not require any further explanation here.
• the expression system according to the invention is suitable for the expression of various heterologous and homologous proteins.
• the expression of homologous proteins is advantageous when the expression of a protein present in the organism used is to be increased, since the promoter according to the invention can greatly improve the amount of protein produced.
• the system is preferably used for the expression of hormones, e.g. Growth hormones and growth factors, immunomodulating factors, e.g. Interferons and interleukins, enzymes e.g. Endopolygalacturonase, reporter genes, e.g. EGFP, or antigens, e.g. Surface antigens from viruses, including S antigens from hepatitis B virus or virus protein 1 from polyoma virus. The latter proteins can be used particularly advantageously as vaccines.
• hormones e.g. Growth hormones and growth factors
• immunomodulating factors e.g. Interferons and interleukins
• enzymes e.g. Endopolygalacturonas
• the expression cassette according to the invention is suitable, inter alia, for yeasts of the strains Kluyveromyces and Saccharomyces and is preferably used in yeast strains of the species Kluyveromyces marxianus var. Marxianus.
• TPI is an intracellular enzyme and catalyzes metabolic processes that normally take place inside the cell. TPI is therefore usually not expected and not found in the medium surrounding the cell. As is to be expected with an enzyme with an intracellular effect, no signal sequence was therefore found.
• TPI has now been detected in a special microorganism, namely Kluyveromyces marxianus, in the cell supernatant.
• signal sequences were searched in the open reading frame, which codes for TPI, but no typical sequences could be found.
• the present invention takes advantage of the property of the KmTPI enzyme to leave the cell.
• Peptides and proteins are naturally removed from the cell if they have a signal sequence. During the translation, this signal sequence creates a section which ensures membrane contact and the passage of the protein to be removed. This mechanism is obviously not available at KmTPI, but the special triose phosphate isomerase from K. marxianus has properties that enable the cell membranes to penetrate. In addition, it was found that a fusion-attached peptide or protein is also released from the cell together with the triose phosphate isomerase.
• KmTPI is not only released from the cell in K. marxianus after expression, but that after transformation of other yeast strains with autonomously replicating plasmids which can express the KmTPI gene, it overexpresses and release of TPI into the culture medium, and that on the other hand it is possible to transfer fusion proteins with TPI, foreign proteins after transcription and translation as fusion proteins into the surrounding cell medium.
• the invention therefore furthermore relates to a process for removing a foreign protein in the form of a fusion product with triose phosphate isomerase, in which a sequence, the at least regulatory sequences and a fusion DNA which codes for triose phosphate isomerase and the desired peptide or protein, are expressed , the fusion protein formed is isolated and the foreign protein is separated.
• the fusion product can be both a hybrid molecule, ie consist of a homologous and a heterologous portion, as well as a fusion protein consisting of two, or possibly more, heterologous parts.
• the TPI part on the foreign protein can be both N-terminal and C-terminal.
• the TPI part at the N-terminal end of the foreign protein is preferred for the removal from the cell.
• the fusion product can then be separated in a manner known per se.
• a DNA sequence coding for a spacer is preferably inserted in a manner known per se between the two DNA sequences coding for the proteins.
• the spacer between the two parts of the molecule should be large enough to enable easy separation and, in a particularly preferred embodiment, provides an interface for enzymes.
• triosephosphate isomerase even removes hydrophobic peptides or proteins from cells which cannot be found in the supernatant or can only be found to a small extent when using conventional signal sequences. This has been shown for the surface antigen of hepatitis B, (Hbs), which cannot be removed from the cell with the usual methods.
• the removal of proteins via the fusion with TPI is therefore particularly suitable for those proteins which, owing to their hydrophobicity or lack of signal peptides, are normally not removed from the cell.
• a fusion product of KmTPI and the desired foreign protein This can be done in the insertion cloning site of the expression cassette described above, e.g. B. a a fusion protein co- dier polynucleotide with N-terminal or C-terminal TPI.
• a DNA sequence coding for a linker is preferably provided between the sequences for the two proteins, in order later to facilitate the cleavage of the fusion product.
• the foreign protein is then carried out of the cell together with it as a fusion product due to the "ability to remove" of KmTPI and can then be obtained in an elegant manner after separation from the cell medium and by cleavage of KmTPI.
• a signal sequence known per se can also be used to remove an expressed protein. This is ligated between the promoter and the foreign gene to be expressed in a manner known per se.
• a signal sequence which is homologous to the organism used is preferably used. Particularly good results were achieved with a combination of the promoter of triose phosphate isomerase, or a part thereof which acts as a promoter, and the signal sequence of the endopolygalacturonase gene from Kluyveromyces marxianus.
• a further improvement in expression and secretion is obtained if the discharging action of TPI is enhanced by the provision of a signal sequence.
• the embodiment described above in which a DNA sequence encoding a fusion product of KmTPI with the desired foreign protein, can therefore also be used in combination with a known signal sequence, as described in the previous section. This combination is also part of the present invention.
• an expression and secretion system which, in operative connection, has the promoter sequence defined above or a part thereof active as a promoter, a sequence active as a terminator, and, between these two sequences, a signal sequence.
• a known sequence is preferably used as the signal sequence. This is the signal sequence of the enzyme endopolygalacturonase (EPG) from K. marxia- nus. The signal sequence of the enzyme EPG from K. marxianus is therefore preferably used.
• the cultivation is carried out in a manner known per se, either the protein is continuously released into the medium in a continuous process and can be continuously obtained from the fermentation broth or the cells are cultivated, harvested and then the protein extracted in a batch process the broth can be obtained.
• the system according to the invention is very variable. For example, only the promoter sequence according to the invention defined above or a part thereof active as a promoter together with other nucleic acid sequences which provide further regulatory sequences and are combined with a heterologous nucleotide sequence.
• the promoter sequence defined according to the invention or a part thereof active as a promoter can be combined with a terminator sequence, for example the one defined above, in order to provide a system which is homologous to Kluyveromyces marxianus and in which the polynucleotide for the protein to be expressed is used , or a system comprising the promoter according to the invention, a signal sequence and a terminator can be combined together with a gene to be expressed, which encodes a desired protein, in order to produce a protein to be released into the culture.
• the promoter sequence according to the invention or a part thereof active as a promoter can be combined with a terminator sequence and a nucleic acid from KmTPI and the desired foreign gene.
• the invention further relates to plasmids which contain expression systems according to the invention, in particular plasmids which contain the promoter, the TPI gene and the terminator of TPI from K. marxianus.
• plasmids which contain the promoter, the TPI gene and the terminator of TPI from K. marxianus.
• Examples are the plasmids R64, R53, R48 and R1 1 which are explained in more detail in FIGS. 1 to 4.
• These plasmids are recombinant plasmids and can be used in the present form for amplifying the expression cassettes and for generating the proteins encoded by the ligated DNA in yeasts.
• the plasmid pD1 which contains the sequence according to SEQ ID No. 1, was in E.coli DH5 ⁇ as the host cell at the German Collection of Microorganisms (DSMZ) on January 4, 2001 with the deposit no. DSM 13973 deposited.
• a system which allows the promising yeast type Kluyveromyces marxianus to be used as a host due to its exceptional physiological performance.
• the system according to the invention is suitable for the expression of RNA, peptides, polypeptides, proteins and hybrid molecules including glycosylated proteins.
• An "expression vector” is a DNA molecule that can be linear or circular and contains a segment that encodes a sequence for a protein or peptide of interest that is operatively linked to regulatory sequences. These regulatory sequences include at least promoter and terminator sequences.
• the expression vector can additionally contain selectable markers and other regulatory elements and must enable the transfer and multiplication in host cells.
• the expression vectors can be replicated autonomously or by integration into the host genome.
• DNA or "polynucleotide” includes polymeric forms of deoxyribonucleotides of any length and any modification in single and double-stranded form.
• secretion vector denotes an expression vector which, in addition to the expression of a polypeptide, also causes the polypeptide to be discharged in the form of a fusion protein or through the signal sequence.
• operatively connected means that the individual segments are arranged in such a way that they serve the intended purpose, i.e. can initiate and terminate transcription and can promote expression or enable expression and secretion.
• the claimed sequences can have further short sequences that do not interfere with the biological activity of the molecule.
• the claimed sequences also include allelic variants of the sequence, i.e. alternative forms of the gene that were created by mutation.
• protein or peptide refers to a molecular chain of amino acids with biological activity.
• polypeptide usually refers to amino acid sequences with up to 200 amino acids, while longer chains are usually referred to as proteins.
• polypeptide is also intended to include proteins or, conversely, the term proteins is also intended to include polypeptides.
• the proteins and / or polypeptides can be modified in vivo or in vitro, e.g. through glycosylation and phosphorylation.
• Foreign DNA is any DNA that is not normally expressed under the control of the TPI promoter.
• Foreign DNA can include genes, parts of genes, fused genes, cDNA or other DNA sequences, as well as DNA, the polypeptides or proteins or also RNA or encoded anti-sense RNA.
• Foreign DNA can also be a reporter gene.
• the foreign DNA can be a naturally occurring, genetically engineered or chemically synthesized sequence or a combination thereof. The nucleotide sequence used and the method of its preparation are not critical.
• the invention further relates to a method for producing a recombinant protein, which is characterized in that a yeast cell is transformed with an autonomously replicating plasmid which comprises an expression cassette according to the invention and a polynucleotide which encodes a foreign protein, the yeast cell under conditions suitable for the expression of the foreign protein, and the protein wins.
• the invention also relates to a method for producing a recombinant protein, which is characterized in that an expression cassette according to the invention is placed in a yeast cell, where the expression cassette is incorporated into the genome of the host cell, the cell is grown and the protein is subsequently obtained.
• the expression cassette according to the invention is particularly preferably used as a module which enables the construction of episomal or integrative expression vectors which contain the regulatory sequences and possibly signal sequences according to the invention.
• the use of the promoter according to the invention in combination with a sequence for a desired protein and with a terminator leads to the expression of the desired protein, in particular in yeast cells.
• the expression of the gene EGFP (enhanced green fluorescent protein) under the control of the CMV (cytomegalovirus) promoter was compared with that of the EGFP gene under the control of the TPI promoter , It was found that the expression under the TPI promoter according to the invention effects about 50 times the expression of the CMV promoter, which shows that the promoter according to the invention leads to an increase in the expression of foreign proteins.
• EGFP enhanced green fluorescent protein
• CMV cytomegalovirus
• Fig. 1. is a schematic representation of an expression cassette (R64) according to the invention, in which a functional part of the Km-TPI maximum promoter (position 21 to 1 1 1 5 corresponding to SEQ ID No. 1), the reading frame of the EGFP gene and a functional part of the Km- TPI terminators (positions 1860 to 21 27 according to SEQ ID No. 1) are operatively connected to one another.
• Fig. 2 the expression cassette R53 according to the invention, in which a functional part of the Km-TPl maximum promoter (position 21 to 1115 corresponding to SEQ ID No. 1), the reading frame of the hepatitis B virus S antigen and a functional part of the Km-TPI terminator (positions 1860 to 21 27 according to SEQ ID No. 1) are operatively connected to one another.
• Fig. 3 the expression cassette R48 according to the invention, in which a functional part of the Km-TPl maximum promoter (position 21 to 1 1 1 5 corresponding to SEQ ID No. 1), the reading frame of the hepatitis B virus S antigen, the reading frame for triose phosphate isomerase without start methionine (position 1 1 1 6 to 1859 according to SEQ ID No. 1) and a functional part of the Km-TPI terminator (positions 1860 to 2127 according to SEQ ID No. 1) are operatively linked.
• a functional part of the Km-TPl maximum promoter position 21 to 1 1 1 5 corresponding to SEQ ID No. 1
• the reading frame of the hepatitis B virus S antigen the reading frame for triose phosphate isomerase without start methionine
• a functional part of the Km-TPI terminator positions 1860 to 2127 according to SEQ ID No. 1
• Fig. 4 the expression cassette R1 1 according to the invention, in which a functional part of the Km-TPl maximum promoter (position 21 to 1 1 12 according to SEQ ID No. 1), the reading frame for triose phosphate isomerase (position 1 1 13 to 1856 according to SEQ ID No. 1) the reading frame of the hepatitis B virus S antigen, and a functional part of the Km-TPI terminator (positions 1857 to 2037 according to SEQ ID No. 1) are operatively linked.
• 5 shows the comparison of the expression of the TPI gene in the K. marxianus parent strain and after transformation with the plasmid pKmarTI, which consists of the sequence corresponding to SEQ ID No. 1 ' and the K.
• a DNA fragment is amplified by PCR using the primers P23 and P36, which consists of the promoter, coding region and terminator of the KmTPI gene and is limited at the ends by artificial restriction enzyme recognition sites for Mlu ⁇
• This fragment is cloned into the Mlu ⁇ Site of the plasmid pSL1 180.
• a restriction recognition site for the restriction enzyme Acl (AACGTT).
• PCR introduces a silent mutation that destroys the Acl site, but does not change the amino acid valine encoded by GTT (AACGTT to AACGTC).
• a site for Acl ⁇ can now be generated by PCR immediately before the start codon, since this is where the nucleotide sequence AAC ATG (AACgtt ATG) is located.
• AACgtt ATG nucleotide sequence AAC ATG
• GTC TAA stop codon of the TPI coding region
• acGTt TAA aacGTt TAA
• the cassettes can be installed in a known manner via M / u ⁇ in corresponding integrative or autonomously replicating vectors and introduced into recipient cells with these.
• Expression units can also be generated on the basis of the regulatory sequences of the KmTPI generally by overlapping PCR reactions using hybrid primers, without Acft interfaces having to be generated in the sequence. Appropriate techniques and PCR regimes are known to the person skilled in the art. The system described in Example 1 is therefore not to be seen exclusively, but only as an example.
• Example 3
• R64, R 53, R48 and R1 1 have been generated by PCR techniques. These cassettes are characterized in more detail by schemes and restriction maps in FIGS. 1 to 4.
• the cassette contains the KmTPI maximal promoter with nucleotides 21 to 1 1 15 of SEQ ID No. 1, the EGFP reading frame and the KmTPI terminator with nucleotides 1860 to 21 27 according to SEQ ID No. 1 (TPIpr-MetEGFP- TPItr).
• the cassette contains the KmTPI maximal promoter with nucleotides 21 to 1 1 1 5 of SEQ ID No. 1 (including Met), the reading frame for hepatitis B virus S antigen and the KmTPI terminator with nucleotides 1860 to 21 27 accordingly Sequence SEQ ID No. 1 (TPIpr-HBS-TPItr)
• the cassette contains the KmTPI maximal promoter with nucleotides 21 to 1 1 1 5 of SEQ ID No. 1 (including Met), the reading frame for hepatitis B virus S antigen at whose C-terminus the reading frame for TPI without methionine is fused and the KmTPI terminator with nucleotides 1860 to 2127 according to sequence SEQ ID NO.1 (TPIpr-HBS-TPI-TPItr).
• Plasmid R1 1 ( Figure 4)
• the cassette contains the KmTPI maximal promoter and the entire reading frame for KmTPI at whose C-terminus is fused with methionine starting with the reading frame for hepatitis B virus S antigen. This ends with its own stop codon (UAG).
• the functional part of the KmTPI terminator used here overlaps with the stop codon of the HBs sequence (TAA ATT AA ATT AGG) and begins with the stop codon UAA at position 1857 in accordance with SEQ ID No. 1. It only comprises 180 base pairs.
• This cassette was clipped over blunt ends in the Sma ⁇ site of corresponding plasmids (TPIpr-TPI-HBS-TPItr).
• This international depository accepts the microorganism referred to under I, which it received on 2 001 - 0 1 - 04 (date of first deposit) '.
• microorganism referred to under I was received by this international depository on (date of first deposit) and an application for the conversion of this first deposit into a deposit under the Budapest Treaty was received on (date of receipt of the request for conversion).

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• 2001
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• 2002
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  • 2002-01-16 US US10/466,758 patent/US20040161841A1/en not_active Abandoned
  • 2002-01-16 EP EP02748300A patent/EP1354052A2/de not_active Withdrawn
  • 2002-01-16 JP JP2002570738A patent/JP2004519239A/ja not_active Withdrawn

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