EP1175494A1 - Beta-hexosaminidase sowie diese kodierende dna-sequenz aus ciliaten und deren verwendung - Google Patents
Beta-hexosaminidase sowie diese kodierende dna-sequenz aus ciliaten und deren verwendungInfo
- Publication number
- EP1175494A1 EP1175494A1 EP00910764A EP00910764A EP1175494A1 EP 1175494 A1 EP1175494 A1 EP 1175494A1 EP 00910764 A EP00910764 A EP 00910764A EP 00910764 A EP00910764 A EP 00910764A EP 1175494 A1 EP1175494 A1 EP 1175494A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleic acid
- sequence
- hexosaminidase
- ciliates
- acid according
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
Definitions
- the invention relates to a nucleic acid coding for a ⁇ -hexosaminidase.
- Bacterial expression systems based on E. coli or B. subtilis are used for the production of recombinant peptides or proteins such as e.g. Insulin, interleukin-2, tissue plasminogen activator, proteases and lipases are used.
- the expression systems are mostly based on the use of genetic elements, such as the lac operon or tryptophan operon.
- the foreign proteins are either produced in "inclusion bodies" within the cell or, when using expression systems based on ⁇ -lactamase genes, in the periplasmic space. The production of recombinant proteins in the surrounding fermentation medium has not been established. In the case of gram-positive bacteria, only cell-specific proteins have so far been almost exclusively incorporated and expressed in expression systems.
- Yeasts such as S. cerevisiae, Kluyveromyces lactis or Pichia pastoris
- CONFIRMATION COPY are also used for the heterologous expression of recombinant proteins, such as human factor Xllla, bovine pro-chymosin or surface antigens.
- the expression systems here are based on shuttle vectors (vector with a yeast and a bacterial component), which are based on the genetic elements of galacto-kinase epimerase, acid phosphatase or alcohol dehydrogenase.
- the recombinant protein is produced in the cytoplasm of the cell.
- yeast's own signal sequences such as that of the alpha factor
- the expressed proteins can also be secreted into the fermentation medium. The glycosylation of secreted proteins takes place according to the "high-mannose" type.
- Mammalia cells such as various rodent cell types (CHO cells, C127 cells) or monkeys (Vero, CV-1 or COS cells) are also used for heterologous expression of recombinant proteins.
- the expression systems here are based on recombinant viruses (BPV vector) or on shuttle vectors. Viral SV40 enhancer / promoter systems or cellular enhancer elements are used to regulate expression.
- the recombinant proteins, such as erythropoietin are secreted into the fermentation medium since the foreign genes usually already have signal sequences and these are understood by the expression system and used for targeting.
- Ciliates such as Tetrahymena are also used for the biotechnological production of glycosylated, extracellular enzymes. Ciliates grow on inexpensive fermentation media using standard fermentation processes. Vectors based on the rDNA elements of the ciliate Tetrahymena are available for the transformation of such ciliates. To the heterologous Expression of bacterial proteins in ciliates, DNA constructs from Tetrahymena genes are used. If suitable genetic elements for regulating transcription, targeting and glycosylation of foreign proteins are available, ciliates are an ideal expression system for the cost-effective production of therapeutic recombinant proteins.
- the cells In order to obtain the recombinant protein, the cells have to be broken open and the denatured, inactive protein has to be folded back for its function. This causes additional costly process steps and lowers the yield of the desired protein.
- the glycosylation which is important for eukaryotic proteins, is completely omitted.
- Gram-positive bacterial expression systems the degradation of the target protein due to high proteolytic activities in the fermentation broth is also problematic.
- yeasts When using yeasts for heterologous expression, the desired target protein is often only produced in the cell from which it has to be removed by cell disruption. As with bacterial expression systems, this causes additional time-consuming and costly process steps. If yeast-specific signal peptides are used, the foreign proteins are not correctly spliced and glycosylated during secretion.
- mammalia cell systems are used for the production of recombinant proteins, the desired proteins are extracellular, correctly spliced and glycosylated in the fermentation medium.
- the disadvantage here is that low expression rate due to incorrect processing and inefficient translation of genes that were introduced into the genome of the production cell line via viral vectors.
- the serum-containing fermentation media for mammalia cells are extremely cost-intensive.
- the fermentation technology for the cell lines sensitive to shear force is complex and also expensive due to the designs for bubble-free aeration. Further problems arise from the high risk of infection of the cell lines by mycoplasmas and viruses. Taken together, the use of mammalia cells for the biotechnological production of recombinant proteins results in very high costs, safety requirements and low yields.
- the object of the invention is to provide a DNA sequence for the expression of secreted proteins from ciliates.
- the DNA sequence should make it possible to transfer heterologous proteins into the fermentation medium after transformation into ciliates in an expression system. This system is also said to achieve a high expression rate of the heterologous protein, which is removed from the cell in large quantities under culture conditions.
- This object is achieved by a system in which a nucleic acid with a sequence with the Seq. ID. No. 1 coding for ß-hexosaminidase is used.
- the DNA sequence according to the invention of the ⁇ -hexosaminidase contains, in particular, a signal and a propeptide, and, if appropriate, further genetic elements for targeting proteins.
- the use of these sequences in a vector makes it possible to transport heterologously expressed proteins from the cell and thus to purify them from fermentation broth without cell disruption.
- FIG. 1 shows a nucleic acid coding for ⁇ -hexosaminidase from ciliates.
- FIG. 2 shows a corresponding expression product of the nucleic acid according to Seq. ID. No. 1. This protein according to Seq. ID. No. 2 is also the subject of the present invention.
- the invention particularly relates to the signal sequence of the protein according to the invention. These are preferably amino acids 1 to 17 of the protein according to the invention.
- the invention also relates to a nucleic acid which codes for the N-terminal fragment. It is preferably a fragment of the nucleic acids according to the invention, in particular with the nucleic acid sequence 1 to 51 according to FIG. 1.
- Another aspect of the invention is the use of a nucleic acid sequence of acidic hydrolases according to the invention or parts thereof for the homologous or heterologous expression of recombinant proteins and peptides, and for homologous or heterologous recombination ("knock-out", "gene replacement”).
- the invention also relates to a method in which the nucleic acid according to the invention coding for ⁇ -hexosaminidase or parts thereof with the enhancers, promoters, operators, origins, terminators, antibiotic resistances or other nucleic acids or DNA fragments or other nucleic acids or DNA fragments which are customary for homologous or heterologous expression or Sequences of any kind of viroids, viruses, bacteria, archezoes, protozoa, fungi, plants, animals or humans can be combined.
- nucleic acid according to the invention or parts thereof is incorporated into a vector, a plasmid, a cosmid, a chromosome or minichromosome, a transposon, an IS element, an rDNA or any other type of circular or linear DNA or RNA.
- nucleic acids which are at least 40% homologous with the nucleic acid according to Seq. ID. No. 1 is.
- the protein according to Seq. ID. No. 2 can be modified without losing its function. So-called conservative exchanges of amino acids can be carried out, for example. For this, e.g. hydrophobic amino acids are interchanged.
- the chromatographic fractions collected were tested with specific 4-nitro-phenyl substrates for acid hydrolases, the acid phosphatase (sPAse) and the ⁇ -hexosaminidase ( ⁇ -Hex), and the fractions with the highest ⁇ -hex activity were combined. These were concentrated by a further Amicon ultrafiltration and buffered in the start buffer for affinity chromatography.
- the chromatographic fractions collected were tested for enzyme activities as described above, the fractions with the highest activity for an acidic hydrolase were combined and buffered in phosphate buffer (PB).
- PB phosphate buffer
- the hydrolase-containing chromatography fractions were combined from a total of eight purifications, concentrated, portioned and frozen until further characterization.
- ⁇ -hexosaminidase-specific PCR primers were created. By comparing the sequences with the already existing ⁇ -hex sequences, it was possible to preselect the primer combinations for the RT-PCR. With this information, the first specific cDNA fragments were amplified and sequenced using RT-PCR from isolated total RNA, the quality of which had previously been checked by Northern hybridization. With the help of these fragments, sequence-specific primers were constructed, which were used for further PCR experiments. Each primer and each partial sequence obtained was checked by database comparison and before further experiments, among other things. checked for possibly overlooked vector sequences. The cDNA sequence was extended to the 5 'and 3' end by 5 'and 3' RACE. The completed sequence of the ⁇ -hex cDNA then served as the basis for further sequence analyzes.
- the sequence of a ⁇ -hexosaminidase from the ciliate thus determined is as shown in FIG. 1.
- the sequence is a total of 1836 base pairs including 5'- and 3'-untranslated regions of the ⁇ -hexosaminidase with an open reading frame ("open reading frame") of 1656 base pairs in length.
- the complementary amino acid sequence is 551 amino acids long and is as shown in Figure 2.
- the sequence of the ⁇ -hexosaminidase has a total of 9 glycosylation sites and contains a signal peptide and a pro-sequence for targeting the enzyme by the sorting mechanism of the cell.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
Claims
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19909189 | 1999-03-03 | ||
DE19909189 | 1999-03-04 | ||
DE19958979 | 1999-12-08 | ||
DE19958979 | 1999-12-08 | ||
PCT/EP2000/001853 WO2000052176A1 (de) | 1999-03-03 | 2000-03-03 | β-HEXOSAMINIDASE SOWIE DIESE KODIERENDE DNA-SEQUENZ AUS CILIATEN UND DEREN VERWENDUNG |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1175494A1 true EP1175494A1 (de) | 2002-01-30 |
Family
ID=26052157
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP00910764A Withdrawn EP1175494A1 (de) | 1999-03-03 | 2000-03-03 | Beta-hexosaminidase sowie diese kodierende dna-sequenz aus ciliaten und deren verwendung |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1175494A1 (de) |
JP (1) | JP2004500017A (de) |
AU (1) | AU781876B2 (de) |
CA (1) | CA2372285A1 (de) |
WO (1) | WO2000052176A1 (de) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060127973A1 (en) * | 2002-03-19 | 2006-06-15 | Marcus Hartmann | Dna sequences of major secreted proteins from the ciliate tetrahymena and use thereof |
DE10214406A1 (de) * | 2002-03-30 | 2003-10-09 | Nutrinova Gmbh | Verfahren und Marker zur einfachen Transformation und Selektion von rekombinanten Protisten |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2127697B1 (es) * | 1997-03-05 | 2000-03-16 | Antibioticos Sau | Promotores de los genes glutamato deshidrogenasa, beta-n-acetilhexosaminidasa y gamma-actina y su utilizacion en sistemas de expresion, secrecion y antisentido de hongos filamentosos. |
WO1998050512A1 (en) * | 1997-05-06 | 1998-11-12 | The Procter & Gamble Company | Laundry and cleaning compositions containing hexosaminidase enzymes |
-
2000
- 2000-03-03 WO PCT/EP2000/001853 patent/WO2000052176A1/de active IP Right Grant
- 2000-03-03 EP EP00910764A patent/EP1175494A1/de not_active Withdrawn
- 2000-03-03 CA CA002372285A patent/CA2372285A1/en not_active Abandoned
- 2000-03-03 AU AU32856/00A patent/AU781876B2/en not_active Ceased
- 2000-03-03 JP JP2000602787A patent/JP2004500017A/ja active Pending
Non-Patent Citations (1)
Title |
---|
See references of WO0052176A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU3285600A (en) | 2000-09-21 |
CA2372285A1 (en) | 2000-09-08 |
JP2004500017A (ja) | 2004-01-08 |
WO2000052176A1 (de) | 2000-09-08 |
WO2000052176A8 (de) | 2002-06-20 |
AU781876B2 (en) | 2005-06-16 |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: CILIAN AG |
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RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: CILIAN AG |
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Effective date: 20040702 |
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