WO2002012503A1 - Production dans la levure pichia pastoris et systeme de purification de l'allergene de recombinaison ole e 1 de l'olea europaea utilise dans le diagnostic et le traitement des allergies - Google Patents

Production dans la levure pichia pastoris et systeme de purification de l'allergene de recombinaison ole e 1 de l'olea europaea utilise dans le diagnostic et le traitement des allergies Download PDF

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Publication number
WO2002012503A1
WO2002012503A1 PCT/ES2001/000287 ES0100287W WO0212503A1 WO 2002012503 A1 WO2002012503 A1 WO 2002012503A1 ES 0100287 W ES0100287 W ES 0100287W WO 0212503 A1 WO0212503 A1 WO 0212503A1
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WIPO (PCT)
Prior art keywords
seq
ole
recombinant
polypeptides
olea europaea
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Ceased
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PCT/ES2001/000287
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English (en)
Spanish (es)
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WO2002012503A8 (fr
Inventor
Rosalia Rodriguez Garcia
Maria Teresa Villalba Diaz
Rafael Monsalve Clemente
Eva Batanero Cremades
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Universidad Complutense de Madrid
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Universidad Complutense de Madrid
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Priority to AU2001272565A priority Critical patent/AU2001272565A1/en
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Publication of WO2002012503A8 publication Critical patent/WO2002012503A8/fr
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention refers to one of the olive pollen allergens (Olea europaea), the Ole e 1 protein, and allergenic epitopes present in the protein.
  • the invention also refers to recombinant DNAs that encode both complete proteins and fragments that include one or more epitopes of the molecule, and homologous Ole e 1 molecules with allergenic capacity in other species, such as Lig v 1 pollen Aligustre (igustrum vulgare) and Syr v 1 of lilac pollen (Syr inga vulgaris)
  • the object of the invention is also the production of Ole e 1, Syr v 1 and Lig v 1 proteins by recombinant technology in yeast Pichia pastoris, which implies the use of the nucleotide sequences SEQ ID NO: 1, 2, 3,, 5, 6, 7, 8 or other nucleotide sequences obtained by mutagenesis of the sequences SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8.
  • the invention provides an effective method of isolation for these proteins with high yield.
  • Type I allergy is a disease that affects more than 20% of the population of industrialized countries. This condition is caused by allergens, present in both organisms and biological products - food, mites, insect poisons, pollens, fungi, mammalian epithelia - and in synthetic materials. In most of the allergenic biological sources, the allergens they are proteins of molecular masses between 5 and 70 Da. Symptoms that arise from allergy, such as rhinitis, conjunctitis, or asthma, are caused by the release of cell mediators, such as histamine, from basophils and mast cells, cells of the immune system. Said release is induced by cross-linking of IgEs antibodies bound to high affinity receptors.
  • the cross-linking of the IgE is caused by the binding of the corresponding allergen, or a fragment thereof, through an IgE epitope (contained in said allergen, or in its fragment).
  • the therapy that is currently being used to treat the allergy involves the hyposensitization of the patient by parental or oral administration of adequate doses of the allergen itself, or related allergoids.
  • an allergenic extract is obtained, through minimal manipulation, of the natural biological source, which implies a very complex mixture of proteins and other substances, in which the allergen - or allergens - can represent a very small part of the total product used.
  • the protocols used for the diagnosis of allergy cases and their subsequent immunotherapy involve the use of allergenic extracts that are often not characterized, or even standardized with respect to the most important allergens they may contain. Often, its administration does not provide a complete diagnosis, especially when the patient's hypersensitivity refers to allergens present in low concentration in said extracts. On the other hand, the immunotherapy performed with these preparations is often ineffective and sometimes causes undesirable side effects that may come to be more serious than the allergic condition that is intended to resolve.
  • An alternative to the use of these extracts is the preparation of mixtures of the most significant allergens, obtained by isolation from their natural source. However, this route presents two important barriers.
  • Olive polynose is one of the most important causes of allergy in Mediterranean countries, where the olive is intensely cultivated. At present, several allergens of olive pollen are known, with Ole e 1 showing the highest incidence among patients allergic to olive trees since it affects more than 70% of them. Ole e 1 is a glycoprotein consisting of 145 amino acids - corresponding to 16.3 kDa- and a carbohydrate fraction of 1.5 kDa [Villalba, M, et al. Eur. J. Biochem. 216, 863-869 (1993)]. The DNA encoding Ole e 1 has been cloned, sequenced and expressed in E. coli, by the inventors [Villalba, M., et al. J. Biol.
  • the homologous proteins of Ole e 1 in alligator pollen (Ligustrum vulgare), Lig v 1, and in lilac pollen (Syringa vulgaris), Syr v 1, have been isolated from their natural source, and have originated a cross-allergenic reaction with Ole e 1, against the sera of patients allergic to olive pollen.
  • the specific cDNA encoding these proteins has been cloned, sequenced, and expressed in E. coli [Batanero E., et al Clin. Exp. Allergy 26, 1401-1410, 1996; Batanero E., et al. Eur. J. Biochem. 221, 187-193, 1994].
  • the present invention relates to the production in Pichia pastoris yeast and purification of the recombinant allergen of Olea europaea Ole e 1.
  • recombinant DNA molecules are obtained that encode polypeptides that exhibit the antigenicity of Ole e 1, an olive allergen (Olea europaea) and other plants (lilac and aligustre) that possess homologous allergens of Ole e 1, as well as for polypeptides containing at least one epitope of Ole e 1 in its structure.
  • the invention has DNA polynucleotide sequences that hybridize under restrictive conditions with those described above - which implies an identity level of at least 60% between their nucleotide sequences - or are derived from them by degeneracy of the genetic code or mutagenesis.
  • the procedure for obtaining the "Olea europaea Ole e 1" recombinant allergen includes expression vectors and host cells that contain a nucleotide sequence such as those described in SEQ No. 1, 2, 3, 4, 5, 6, 7, encoders of Ole e 1, homologous proteins or fragments thereof.
  • polypeptides are obtained that possess the antigenic activity of the Ole e 1 allergen of olive tree or of its fragments, as well as of homologous Ole e 1 allergens - mainly in species related to the olive tree, such as all Oleaceae - that, given the structural similarity, at least 60%, have cross allergenic reactivity with Ole e 1 or with a part of it.
  • These polypeptides may contain the antigenic sequence linked to other polypeptides (for example as fusion proteins), or have been chemically or enziically modified.
  • polypeptides The methods of preparing these polypeptides involve their recombinant production from the aforementioned polynucleotide molecules, in a eukaryotic cell culture system containing the expression vectors described as carriers of those A. Once the polypeptide molecule with antigenic activity of Ole e 1 or its epitopes is produced, it is isolated from the extracellular medium of the culture in soluble form by penetrability and ion exchange chromatography, as described in detail below.
  • the objects obtained through the procedure is the ability to bind IgE antibodies of the serum of patients allergic to olive, sensitive to Ole e 1 or their antigenic segments.
  • the procedure described in this invention produces protein levels of an order of greater magnitude: tens of milligrams of protein (about 60) per liter of culture.Finally, with the process object of the invention, Ole e 1 -a will be available olive pollen allergen of the utmost clinical importance - immunologically active, in addition to antigenic fragments of this allergen, and homologous proteins of it, which will be used in the "in vivo” and “in vitro” tests to be carried out for the faithful hypers diagnosis Ensibility to this pollen, and other pollen related phylogenetically with it. They may also be used in allergen preparations that are used to carry out the corresponding immunotherapy for the treatment of olive pollen allergy.
  • Protein extracts obtained from pollen are currently used for the diagnosis and therapy of olive pollen allergy. This implies a scarce reproducibility and a high content of contaminating molecules, of protein and non-protein origin, which can cause adverse side effects in treated patients.
  • the availability of homogeneous molecules obtained by recombinant DNA techniques, in unlimited quantities, perfectly quantifiable and standardized, will considerably reduce all the aforementioned inconveniences. This technology allows these molecules to be available and in addition to peptides or modified forms thereof containing at least one of the allergenic epitopes.
  • Olea europaea Ole e 1 recombinant allergen
  • the Ole e 1, Syr v 1 and Lig v 1 allergens purified from olive pollen (Olea europaea) (Villalba, M., et. al. Eur. J. Biochem. 216, 863-869, 1993), of lilac (Syringa vulgaris) (Batanero E., et al. J. Biochem. 211, 187-193, 1994) and of aligustre (Ligustrum vulgare) (Batanero E., et al. Clin. Exp. Allergy 26, 1401-1410, 1996).
  • the pollens were supplied by the Allergon AB commercial house.
  • the knowledge of the amino acid sequence of the amino and carboxyl terminal zone of Ole e 1 allowed the design of the oligonucleotides with which its cloning was addressed by PCR and expression.
  • the procedure object of this invention is carried out by the following phases:
  • RNAs from different pollens used corn and birch
  • olive tissues used seed, leaves and fruits
  • an additional initial stage was introduced in which they were macerated in the presence of liquid nitrogen until a fine and homogeneous powder was obtained. From 5 ⁇ g of total RNA, double stranded DNA synthesis was carried out using the cDNA synthesis kit (Clontech).
  • 5 'ATCATTRTTNGGNGGRTACATNCC3' corresponding to the amino and carboxyl terminal sequence of the protein, respectively, were dissolved in a standard PCR mixture (250 ⁇ M dNTPs, standard buffer and 50 pmoles of the primers). After a first stage of denaturation at 95 ° C for 15 min, 30 cycles were carried out that each included a stage of denaturation at 94 ° C for 1 min, hybridization at 42 ° C for 1 min 30 s and extension at 72 ° C for 2 min in the presence of Taq DNA polymerase (US. Biochemical Corp.).
  • oligonucleotides used in this reaction were OL3 5 'CCGGGATCCGAARGAYGTNCCNCA 3' and OL4
  • amino acid sequences of the proteins encoded by the clones corresponding to the specific DNA of Ole e 1 are SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11, those corresponding to Syr v 1, SEQ ID NO 12, SEQ ID NO 13 and SEQ ID NO 14 and those corresponding to Lig v 1, SEQ ID NO 15 and SEQ ID NO 16.
  • the DNA coding sequence was linked to the expression plasmid pPIC9 (Invitrogen Corp.).
  • the cells used were from the GS115 strain of Pichia pastoris.
  • the induction was carried out by 0.5% methanol for four days, the cells growing at 30 ° C.
  • the cells were pelleted by centrifuging at 5000 xg and the supernatants were collected and a sample thereof was applied on a 15% polyacrylamide gel in the presence of SDS. A unique 20.5 kDa band was detected by staining the gels with Coomassie Blue.
  • the protein is recognized by the natural Ole e 1 specific polyclonal antibody (1: 5000 dilution), by several monoclonal antibodies and by sera from patients allergic to Ole e 1. Protein purification was carried out by two stages, one Ion exchange chromatography in DEAE-cellulose and a gel filtration in Sephadex G-75, after which the recombinant allergen is obtained at a concentration of 15 to 35 mg / ml and with a purity level of 99%. Structural characterization studies (near and far UV and dichroism spectra and fluorescence spectra) and immunology (with individual sera of allergic patients and with 9 monoclonal antibodies) and in all cases the recombinant proteins behaved in a manner equivalent to that of the natural allergen.

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Biotechnology (AREA)
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Abstract

L'invention concerne la production dans la levure Pichia pastoris et un système de purification de l'allergène de recombinaison Ole e 1 de l'Olea europaea utilisé dans le diagnostic et le traitement des allergies. L'ADN de recombinaison codant pour les allergènes Ole e 1 du pollen de l'olivier (Olea europaea), Syr v 1 du pollen du lilas (Syringa vulgaris) et Lig v 1 du pollen du troène (Ligustrum vulgare) est cloné et exprimé. Ces allergènes possèdent une grande similitude structurelle entre eux et avec d'autres allergènes du pollen d'autres Oléacées. Les protéines de recombinaison produites dans la levure Pichia pastoris sont isolées à un haut rendement, sont correctement pliées et présentent des propriétés immunologiques équivalentes à celles des allergènes naturels, ce qui permet de les utiliser dans des protocoles de diagnostic et d'immunothérapie.
PCT/ES2001/000287 2000-07-28 2001-07-19 Production dans la levure pichia pastoris et systeme de purification de l'allergene de recombinaison ole e 1 de l'olea europaea utilise dans le diagnostic et le traitement des allergies Ceased WO2002012503A1 (fr)

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AU2001272565A AU2001272565A1 (en) 2000-07-28 2001-07-19 Production in pichia pastoris yeast and system for purifying the recombinant allergen of olea europaea ole e 1 for utilization in the diagnosis and treatment of allergies

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ESP200001919 2000-07-28
ES200001919A ES2209563B1 (es) 2000-07-28 2000-07-28 Produccion en la levadurapipchia pastoris y sistema de purificacion del alergeno recombinante de olea europaea ole e 1 para uso en diagnosis y tratamiento de alergias.

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WO2002012503A8 WO2002012503A8 (fr) 2002-09-19

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080837A1 (fr) * 2002-03-22 2003-10-02 Universidad Complutense De Madrid Rectorado Production d'allergene recombinant de chenopodium album che a 1 et systeme d'isolement et de purification
ES2199050A1 (es) * 2002-03-22 2004-02-01 Univ Completense De Madrid Produccion del alergeno recombinante de "chenopodium album" che a 1 y sistema de aislamiento y purificacion.
WO2007140505A2 (fr) 2006-06-09 2007-12-13 Biomay Ag Support de vaccin
ES2293749A1 (es) * 2003-06-06 2008-03-16 Universidad Complutense De Madrid Variante recombinante del alergeno frae 1 del polen de fresno "fraxinus excelsior", produccion en la levadura pichia pastoris y aplicaciones.

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110567787A (zh) * 2019-09-12 2019-12-13 浙江工商大学 一种快速纯化血清中IgG和IgE的方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BATANERO E. ET AL.: "Isolation and characterization of an olive allergen-like protein from lilac pollen", EUR. J. BIOCHEM., vol. 221, 1994, pages 187 - 193, XP002953354 *
BATANERO E. ET AL.: "Isolation, cDNA cloning and expression of Lig v1, the major allergen from privet pollen", CLINICAL AND EXPERIMENTAL ALLERGY, vol. 26, 1996, pages 1401 - 1410, XP002953364 *
HUECAS S. ET AL.: "Production and detailed characterization of biologically active olive pollen allergen Ole e I secreted by the yeast Pichia pastoris", EUR. J. BIOCHEM., vol. 261, April 1999 (1999-04-01), pages 539 - 545, XP002953352 *
VILLALBA M. ET AL.: "Cloning and expression of Ole e I, the major allergen from olive tree pollen", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 21, 27 May 1994 (1994-05-27), pages 15217 - 15222, XP002953353 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003080837A1 (fr) * 2002-03-22 2003-10-02 Universidad Complutense De Madrid Rectorado Production d'allergene recombinant de chenopodium album che a 1 et systeme d'isolement et de purification
ES2199050A1 (es) * 2002-03-22 2004-02-01 Univ Completense De Madrid Produccion del alergeno recombinante de "chenopodium album" che a 1 y sistema de aislamiento y purificacion.
ES2293749A1 (es) * 2003-06-06 2008-03-16 Universidad Complutense De Madrid Variante recombinante del alergeno frae 1 del polen de fresno "fraxinus excelsior", produccion en la levadura pichia pastoris y aplicaciones.
ES2293749B1 (es) * 2003-06-06 2009-03-16 Universidad Complutense De Madrid Variante recombinante del alergeno frae 1 del polen de fresno "fraxinus excelsior", produccion en la levadura pichia pastoris y aplicaciones.
WO2007140505A2 (fr) 2006-06-09 2007-12-13 Biomay Ag Support de vaccin
WO2007140505A3 (fr) * 2006-06-09 2008-03-20 Biomay Ag Support de vaccin
US9296828B2 (en) 2006-06-09 2016-03-29 Biomay Ag Vaccine carrier

Also Published As

Publication number Publication date
AU2001272565A1 (en) 2002-02-18
ES2209563B1 (es) 2005-10-01
ES2209563A1 (es) 2004-06-16
WO2002012503A8 (fr) 2002-09-19

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