WO2004065593B1 - Recombinant bovine pepsin and pepsinogen produced in prokaryotic and eukaryotic cells - Google Patents
Recombinant bovine pepsin and pepsinogen produced in prokaryotic and eukaryotic cellsInfo
- Publication number
- WO2004065593B1 WO2004065593B1 PCT/ES2004/070002 ES2004070002W WO2004065593B1 WO 2004065593 B1 WO2004065593 B1 WO 2004065593B1 ES 2004070002 W ES2004070002 W ES 2004070002W WO 2004065593 B1 WO2004065593 B1 WO 2004065593B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequence
- amino acid
- cect
- nucleotide sequence
- seq
- Prior art date
Links
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6478—Aspartic endopeptidases (3.4.23)
- C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a system for the production of recombinant bovine pepsin, comprising the construction, using different cDNAs and synthetic sequences, of a cDNA which can be expressed in microbial, prokaryotic or eukaryotic cells, and the conditions for the production of said pepsin in micro-organisms. Said enzyme can be used for the same applications as the native bovine enzyme, but without the sanitary risk associated with the use of animal tissues.
Claims
1. Un ácido nucleico aislado cuya secuencia de nucleótidos se selecciona entre: a) la secuencia de nucleótidos mostrada en la SEQ. ID. NO: 1 ; b) una secuencia de nucleótidos que comprende la secuencia de nucleótidos comprendida entre el nucleótido 139 y el micleótido 1.119 de la secuencia de nucleótidos mostrada en la SEQ. ID. NO: 1, que codifica la pepsina bovina madura cuya secuencia de aminoácidos está comprendida entre el aminoácido +1 y el aminoácido +326 de la secuencia de aminoácidos mostrada en la SEQ. ID, NO: 2; c) una secuencia de nucleótidos como la definida en a) que comprende unos cambios conservativos en dicha secuencia, en donde dichos cambios consisten en mutaciones, inserciones o deleciones introducidas en dicha secuencia definida en a), conservando la secuencia resultante la funcionalidad de codificar la pepsina bovina madura; y d) una secuencia de nucleótidos como la definida en b) que comprende unos cambios conservativos en dicha secuencia, en donde dichos cambios consisten en mutaciones o inserciones introducidas en dicha secuencia definida en b), conservando la secuencia resultante la funcionalidad de dicha secuencia de nucleótidos definida en b) de codificar la pepsina bovina madura.1. An isolated nucleic acid whose nucleotide sequence is selected from: a) the nucleotide sequence shown in SEQ. ID. NO: 1; b) a nucleotide sequence comprising the nucleotide sequence comprised between nucleotide 139 and the micleotide 1.119 of the nucleotide sequence shown in SEQ. ID. NO: 1, which encodes mature bovine pepsin whose amino acid sequence is comprised between the amino acid +1 and the amino acid +326 of the amino acid sequence shown in SEQ. ID, NO: 2; c) a nucleotide sequence as defined in a) comprising conservative changes in said sequence, wherein said changes consist of mutations, insertions or deletions introduced in said sequence defined in a), the resulting sequence retaining the functionality of coding the mature bovine pepsin; and d) a sequence of nucleotides as defined in b) comprising conservative changes in said sequence, wherein said changes consist of mutations or insertions introduced in said sequence defined in b), the resulting sequence retaining the functionality of said nucleotide sequence defined in b) of coding mature bovine pepsin.
2. Acido nucleico según la reivindicación 1, cuya secuencia de ime ótidos se selecciona entre: a) la secuencia de nucleótidos mostrada en la SEQ. ID. NO: 1; y b) la secuencia de nucleótidos comprendida entre el nucleótido 1 9 y el nucleótido 1.119 de la secuencia de nucleótidos mostrada en la SEQ. ID. NO: 1.2. Nucleic acid according to claim 1, whose sequence of imides is selected from: a) the nucleotide sequence shown in SEQ. ID. NO: 1; and b) the nucleotide sequence comprised between nucleotide 1 9 and nucleotide 1119 of the nucleotide sequence shown in SEQ. ID. NO: 1.
3. Un vector que comprende un ácido nucleico según cualquiera de las reivindicaciones 1 ó 2.3. A vector comprising a nucleic acid according to any of claims 1 or 2.
4. Vector según la reivindicación 3, caracterizado porque es el plásmido pBPOl depositado en la Colección Española de Cultivos Tipo (CECT) con el número de acceso CECT 5722.Vector according to claim 3, characterized in that it is the pBPOl plasmid deposited in the Spanish Type Culture Collection (CECT) with the access number CECT 5722.
5. Vector según la reivindicación 3. caracterizado porque es un vector de expresión.Vector according to claim 3, characterized in that it is an expression vector.
6. Vector según la reivindicación 5, seleccionado entre el plásmido pBP03 depositado en la CECT con el número de acceso CECT 5723 y el plásmido pBP05 depositado en la CECT con el número de acceso CECT 11778. 6. A vector according to claim 5, selected from plasmid pBP03 deposited in the CECT with the access number CECT 5723 and the plasmid pBP05 deposited in the CECT with the access number CECT 11778.
7. Una célula Iransformada con un vector según cualquiera de las reivindicaciones 3 a 6.7. A cell Iransformed with a vector according to any of claims 3 to 6.
8. Célula según la reivindicación 7, transformada con un vector según cualquiera de las reivindicaciones 5 ó 6. The cell according to claim 7, transformed with a vector according to any of claims 5 or 6.
9. Célula según cualquiera de las reivindicaciones 7 ú 8. seleccionada entre células de bacteria y células de levadura.9. Cell according to any of claims 7 or 8. selected from bacterial cells and yeast cells.
10. Célula según la reivindicación 9, seleccionada entre células de Esch&richia cali y células de Saccharo yces cerevisiae.10. The cell according to claim 9, selected from Esch & richia cali cells and Saccharo yces cerevisiae cells.
11. Un procedimiento para la producción de pepsinógeπo bovino recombinante que comprende cultivar una célula transformada según la reivindicación 8 bajo condiciones que permiten la producción de pepsinógeno bovino.11. A process for the production of recombinant bovine pepsinogeo, which comprises culturing a transformed cell according to claim 8 under conditions that allow the production of bovine pepsinogen.
12. Procedimiento según la reivindicación 11, en el que dicha célula transformada es una célula de Eschβrichia CQIΪ transformada con el plásmido pBP03 depositado en la CECT con el número de acceso CECT 5723, The method according to claim 11, wherein said transformed cell is a Eschβrichia CQIΪ cell transformed with the plasmid pBP03 deposited in the CECT with the access number CECT 5723,
13. Procedimiento según la reivindicación 11, en el que dicha célula transformada es una célula de Saccharomyces c&evisiae transformada con el plásmido pBPOS depositado en la CECT con el número de acceso CECT 11778.The method according to claim 11, wherein said transformed cell is a Saccharomyces c & evisiae cell transformed with the plasmid pBPOS deposited in the CECT with the access number CECT 11778.
14. Procedimiento según cualquiera de las reivindicaciones 11 a 13 que comprende- ademas, el procesamiento del pepsinógeno bovino recombinante obtenido para obtener pepsina bovina recombinante.14. Process according to any of claims 11 to 13 further comprising the processing of the recombinant bovine pepsinogen obtained to obtain recombinant bovine pepsin.
15. Procedimiento según la reivindicación 14, en el que dicho procesarπieαto comprende la acidificación de una solución que contiene pepsinógeno bovino recombinante.The process according to claim 14, wherein said process pipette comprises the acidification of a solution containing recombinant bovine pepsinogen.
16. Una proteína recombinante, seleccionada entre a) pepsinógeno bovino recombinante cuya secuencia de aminoácidos se muestra en la SEQ. ID. NO: 2, b) pepsina bovina madura recombinante cuya secuencia de aminoácidos está comprendida entre el aminoácido +1 y el aminoácido +326 de la secuencia de aminoácidos mostrada en ía SEQ. ID. NO: 2, y c) una protei.ua como la definida en a) o en b) que comprende, al menos, la secuencia de aminoácidos de la pepsina bovina madura o cambios conservativos en dicha secuencia que conserven su funcionalidad, en donde dichos cambios consisten en mutaciones, inserciones o deleciones, obtenida según el procedimiento de cualquiera de las reivindicaciones 11 a 15. 16. A recombinant protein, selected from a) recombinant bovine pepsinogen whose amino acid sequence is shown in SEQ. ID. NO: 2, b) recombinant mature bovine pepsin whose amino acid sequence is comprised between the amino acid +1 and the amino acid +326 of the amino acid sequence shown in SEQ. ID. NO: 2, and c) a protein as defined in a) or b) comprising, at least, the amino acid sequence of mature bovine pepsin or conservative changes in said sequence that retain their functionality, wherein said changes they consist of mutations, insertions or deletions, obtained according to the method of any of claims 11 to 15.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200300179A ES2247872B1 (en) | 2003-01-24 | 2003-01-24 | PEPSINA AND PEPSINOGENO RECOMBINANT BOVES PRODUCED IN PROCEDURAL AND EUCHARIOT CELLS. |
| ESP200300179 | 2003-01-24 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2004065593A1 WO2004065593A1 (en) | 2004-08-05 |
| WO2004065593B1 true WO2004065593B1 (en) | 2004-09-16 |
Family
ID=32749102
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/ES2004/070002 WO2004065593A1 (en) | 2003-01-24 | 2004-01-21 | Recombinant bovine pepsin and pepsinogen produced in prokaryotic and eukaryotic cells |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2247872B1 (en) |
| WO (1) | WO2004065593A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114365792A (en) | 2014-11-11 | 2022-04-19 | 克莱拉食品公司 | Methods and compositions for generating ovalbumin |
| JP2022539879A (en) | 2019-07-11 | 2022-09-13 | クララ フーズ カンパニー | Protein composition and consumable products thereof |
| US10927360B1 (en) | 2019-08-07 | 2021-02-23 | Clara Foods Co. | Compositions comprising digestive enzymes |
-
2003
- 2003-01-24 ES ES200300179A patent/ES2247872B1/en not_active Expired - Fee Related
-
2004
- 2004-01-21 WO PCT/ES2004/070002 patent/WO2004065593A1/en active Search and Examination
Also Published As
| Publication number | Publication date |
|---|---|
| ES2247872B1 (en) | 2007-05-01 |
| ES2247872A1 (en) | 2006-03-01 |
| WO2004065593A1 (en) | 2004-08-05 |
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