ES2255340A1 - Ole e 10 allergen isolation and purification technique consists of recombinant production in yeast and treatment with olive pollen for use in immunotherapy - Google Patents

Abstract

The Ole e 10 allergen isolation and purification technique comprises recombinant production of the protein in Pichia pastoris with isolation based on purified Olea europaea pollen. Also claimed is apparatus implementing the process which produces protein equivalent to the natural allergen in immunological properties, for use in immunotherapy.

Description

Production, isolation and purification system of the Ole e 10 allergen.

Object of the invention

The present invention, as expressed in the description of this specification, refers to one of the olive pollen allergens ( Olea europaea ), the Ole e 10 protein, and allergenic epitopes present in the protein. The invention also refers to recombinant DNAs encoding both the complete protein and fragments that include at least one epitope of the molecule, as well as homologous Ole e 10 molecules with allergenic capacity in other related and unrelated species. Specifically, the object of the invention is the production of the Ole e 10 protein, by recombinant technology in the yeast Pichia pastoris , which involves the use of the nucleotide sequence characterized by SEQ ID NO: 1 or other nucleotide sequences obtained by mutagenesis of the sequence SEQ ID NO: 1.

The invention also provides an effective method. of isolation for both the recombinant allergen and the natural obtained from olive pollen.

State of the art

Type I allergy is a disease that affects more than 20% of the population of industrialized countries (Kay, A.B. (1997) Allergy and allergic diseases, Blackwell Science, Oxford, UK). This condition is caused by allergens, present in both organisms and biological products -foods, mites, insect poisons, pollens, fungi, epithelia of mammals-, as in synthetic materials. In most of the allergenic biological sources, the allergens are proteins of molecular masses between 5 and 70 kDa. The symptoms that are derived from allergy, such as rhinitis, conjunctivitis, or asthma, are caused by the release of cell mediators, such as histamine, from basophils and mast cells, cells of the immune system. This release is induced by the cross-linking of IgEs antibodies bound to high receptors affinity Fc \ epsilonRI, which are in turn anchored to basophils and mast cells. The cross-linking of IgE is caused by the binding of the corresponding allergen, or a fragment yours, through an IgE epitope (contained in said allergen, or in its fragment). The therapy that is currently being used to treating allergy involves patient hyposensitization by parental or oral administration of doses suitable of the allergen itself, or related allergoids. In the almost all cases an extract is administered allergen obtained, by minimal manipulation, from the source natural biological, which implies a very complex mixture of proteins and other substances, in which, the allergen -or allergens- it can represent a tiny part of the total product used.

In effect, at present, the protocols used for the diagnosis of allergy cases and their subsequent immunotherapy involve the use of allergenic extracts that frequently they are not characterized, not even standardized regarding the most important allergens they can contain. TO 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 (Valenta, R .; Lidholm, J .; Niederberger, V .; Hayek, B .; Kraft, D. and Grondlund, H. (1999) Clin. Exp. Allergy 29, 896-904). On the other hand, immunotherapy performed with these preparations it is frequently ineffective and originates in occasions undesirable side effects that can become more serious than the allergic condition that is intended solve. An alternative to the use of these extracts is the preparation of mixtures of the most significant allergens, obtained by isolation from its natural source (Rodríguez, R .; Villalba, M .; Monsalve, R.I. and Batanero, E. (2001) Int. Arch. Allergy Immunol. 125, 185-195). However, this Via presents two important barriers. On the one hand, it implies a good  knowledge of the allergenic components of the material that causes allergy, at least all of its main allergens, and This data is often not available. On the other hand, the Isolation process of known allergens, from biological material, it is often difficult and does not provide necessary quantities or the quality required for later employment, mainly due to the low levels at which it's found. All these problems are accentuated when the material Allergenic is the pollen of a plant species. The large number of pigments, carbohydrates, lipids and insoluble components make Very difficult to handle. Needless to say, your availability is also very low and high economic cost, due to the difficulty of its collection. For all the above, it it makes necessary the design of procedures for the production of allergens to be used in diagnostic and immunotherapy protocols  of allergy The production of recombinant DNA is predicted as the more reproducible and effective way to obtain polypeptides well-defined allergens, not only for clinical use, but even for research

Explanation of the invention.

Production of the recombinant allergen of Olea europaea Ole e 10 in the yeast Pichia pastoris , and isolation and purification system.

The present invention relates to the isolation method used for the purification of the Ole e 10 -allergen allergen of olive pollen of increasing clinical importance, especially in certain geographical areas, reaching a prevalence of 55% in patients allergic to olive pollen in some regions of Andalusia - from olive pollen ( Olea europaea ). More specifically it comprises obtaining, by cloning, recombinant DNA molecules that encode polypeptides with the same or similar antigenic properties as the allergen from olive pollen, or polypeptides that possess at least one epitope of the Ole e 10 allergen. The invention also includes the complete sequence of the cDNA of the Ole e 10 allergen and the complete sequence deduced from the
allergen

The process object of the invention allows carry out the purification of the Ole e 10 allergen from pollen olive from a protein extract of olive pollen by three chromatographic stages, two penetrability chromatographs and one of HPLC, as described in detail below.

The invention has sequences DNA polynucleotides that hybridize, under restrictive conditions, with those described above - which implies a level of identity of at  minus 60% between their nucleotide sequences-, or they are derived from them by degeneracy of the genetic code or mutagenesis

The Olea europaea Ole e 10 recombinant allergen cloning procedure includes cloning vectors and host cells that contain a nucleotide sequence as described in SEQ ID NO: 1, Ole e 10 coding, homologous proteins or fragments thereof.

In this way the sequence is obtained nucleotide that encodes the complete allergen and those nucleotide fragments that have at least one sequence that encodes an antigenic determinant of olive Ole e 10 allergen, as well as homologous allergens of Ole e 10 - mainly in species related to the olive tree- which, given the similarity structural, have allergenic cross reactivity with Ole e 10 or with a part of it.

These polypeptides may contain the sequence. antigen linked to other polypeptides (for example as proteins of fusion), have been chemically synthesized or have been obtained by proteolytic digestions and chemical modified or enzymatically.

The insertion of said sequence into vectors of expression in eukaryotic host organisms allows to have of recombinant constructs that can be used for recombinant allergen production.

Once the polypeptide molecule is produced with Ole e 10 antigenic activity amid minimal growth, it is isolated from the extracellular medium of the culture in soluble form by anion exchange chromatography and penetrability.

Recombinant products obtained through  procedure object of the invention have the ability to serum IgE antibody binding of olive allergic patients, sensitive to Ole e 10.

Finally, with the method object of the invention, immunologically active Ole e 10 is available, in addition to antigenic and allergenic fragments of this allergen, and homologous proteins thereof, which serve to be incorporated into the "in vivo" and " tests " in vitro " to be carried out for the faithful diagnosis of hypersensitivity to this pollen, and to 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 allergy to olive pollen.

For the diagnosis and therapy of allergy to olive pollen protein extracts are currently used obtained from pollen. This implies a scarce reproducibility and a high content of pollutant molecules, of Protein and non-protein origin, which may cause effects Adverse side effects in treated patients. The dispose of homogeneous molecules obtained by DNA techniques recombinant, in unlimited quantities, perfectly quantifiable and standardized, would significantly lower all The inconveniences cited. This technology allows to have these molecules and, in addition, peptides or modified forms of same that contain at least one of the allergenic epitopes.

Description of the figures

Figure 1.- Purification of Ole e 10. (A) and (B) elution profiles of gel filtration chromatographs in columns of Sephadex G-75 (35 x 750 mm) and Sephadex G-50 (15 x 530 mm), respectively. The extract of Olive pollen obtained as described below was applied to the first column and the fractions corresponding to molecules of molecular mass less than 15kDa were collected, lyophilized and applied to the second column. (C) Profile of elution in a reverse phase C-18 column in HPLC using a gradient of acetonitrile in 0.1% TFA. The fractions that contain the purified protein are shaded in the figure. (D) PAGE / SDS analysis of the different stages of purification: p line, pollen extract; lines a, b and c, protein obtained in mixing shaded fractions of elution profiles A), B) and C), respectively; m line, molecular weight patterns known. The samples were stained with Coomassie blue or transferred to nitrocellulose membranes and immunostained with a mixture of sera from patients allergic to olive pollen.

Figure 2.- cDNA coding for Ole e 10 (uppercase) and deduced amino acid sequence. The sequence of cDNA of the 3 'and 5' non-coding regions are included in the Figure with lowercase letters. The processing site is indicated by an arrowhead. The DNA sequence is available on the GenBank / EMBL under the access number of AY082335.

Embodiment of the invention

The present invention relates to a recombinant DNA encoding epitopes of the Olea europaea Ole e 10 allergen, to the recombinant production, in Pichia pastoris yeast, of the polypeptides encoded by said DNA and to the isolation of the natural allergen from olive pollen . It also refers to the application of both molecules to the diagnosis and therapy of this immune disorder.

The procedure object of this invention is performs through the following phases:

Total RNA isolation

Total RNA was isolated from olive pollen  following the protocol published by Ullrich et al. [Science 196, 1313-1318, 1977]. In this isolation it started from 0.5 g of pollen that was homogenized in a 0.1 M buffer of Tris-HC1, pH 7.5 containing thiocyanate 4M guanidinium, 0.5% sodium lauryl sarcosinate and 0.14 M 2-mercaptoethanol, using a homogenizer Polytron This suspension was centrifuged at 5000 xg at 20 ° C in a Sorvall centrifuge. Subsequently the supernatant was subjected to a 5.7 M CsCl gradient centrifugation in 0.01 M EDTA at 40,000 rpm on a SW 60 rotor (Beckman) for 12 hours. the same procedure was carried out to isolate other RNAs of different used pollens (aligustre, ash, lilac) and with the different olive tissues used (stem, leaves and fruits). With these last one an additional initial stage was introduced in which they were macerated in the presence of liquid nitrogen until obtaining a powder Fine and homogeneous. From 5 µg of total RNA was carried out cDNA synthesis using the SMART II synthesis kit of cDNA (Clontech).

Cloning of Ole e 10 based on PCR techniques

In the cloning procedure of the recombinant allergen of Olea europaea Ole e 10, a tryptic digestion (weight ratio 1: 1000) of the protein was carried out to obtain internal amino acid sequences of the protein because obtaining the sequence N -terminal through Edman's Degradation was failed because it was blocked.

After sequencing of any of the obtained peptides (AHASY AMNSWYQ SK), the sequence (AMNSWYQ) was used to design the degenerated oligonucleotide ("antisense"), OLE10B: 5'-YTGRTACCANGARTTCAT
NGC-3 'which was used, together with the first unspecific UPM: 5'-CTAATACGACTCACTATAGGGC-3' and using the cDNA as a template, to amplify by PCR the partial sequence coding (1-92) and non-coding 5 'corresponding to the cDNA of Ole e 10, including the signal peptide. The sequences obtained subsequently allowed to obtain the N-terminal sequence, Met Arg Gly Thr Ala Gly Val, which served as the basis for the design of a non-degenerated oligonucleotide, OLE10F: 5'-ATGCGAGGAACCGCAGGTGTG-3 '. Subsequent amplification with the oligonucleotides, OLE10F and UPM, and their cloning allowed obtaining the complete coding nucleotide sequence and the 3 'non-coding sequence. The template and primers of the PCR reactions were dissolved in a standard PCR mixture (250 µM dNTPs, standard buffer and 50 pmoles of the primers). After a first stage of denaturation at 95 ° C for 5 min, 25 cycles were carried out at hybridization temperatures of 45 ° C in the first stage and 68 ° C in the second in the presence of Advantage DNA polymerase II (Clontech) . The reaction was electrophoresed in 1.5% agarose gels and the DNA fragments were purified using the Magic PCR Prep kit (Promega) and inserted into the linearized plasmid pCR2.1 and with a terminal T at the 5 'ends. With this construction, TOP10 competent cells were transformed according to the protocol of the TOPO TA cloning kit (Invitrogen), then the recombinants were selected and the sequencing of some of the selected clones was carried out, obtaining sequences of this allergen. In a final PCR stage the DNA sequence encoding the region corresponding to the mature protein (Ser22-Ser123) was amplified, and using the non-degenerated oligonucleotides NT10: 5'-cgtctcgagaaaagaTCTTCGTCGCCCGTCCCA-3 'for the N-terminal sequence and CT10 : 5'-cgtccgcggTCAAGAGAGGAGAATGAGCATGA-3 'and CT10-2: 5'-cgtccgcggTCAAGAGAGGAATGAGCATGACCCCTGACT-3' for the C-terminal and C-terminal sequence with a Gln instead of the Asn116, possible N-glycosylation site of the protein, respectively .

Recombinant production

For the production of the recombinant form of this protein, the DNA coding sequence was linked to the expression plasmid pPICZα-A (Invitrogen Corp.). The cells used were from the KM71 strain of Pichia pastoris . The induction was carried out by 0.5% methanol for five days, the cells growing at 30 ° C in minimal yeast growth medium. The cells were pelleted by centrifuging at 5000 xg, the supernatants were collected and a sample thereof was applied on a 15% polyacrylamide gel in the presence of SDS. A unique 10 kDa band was detected by staining the gels with Coomassie Blue. The protein is recognized by sera from specific allergic patients of natural Ole e 10 (1:10 dilution). The purification of the proteins was carried out by lyophilization of the extracellular medium of the yeast and the application of two chromatographic stages, one of phonic exchange in DEAE-cellulose and one of penetrability in Sephadex G-50, obtaining after the latter the allergen with a degree of purity of 99%. Structural characterization studies (far UV spectra, mass spectrometry) and immunology (with individual sera of allergic patients) were carried out with them and in all cases the recombinant proteins behaved in an equivalent manner to that of the allergen natural.

Isolation of Ole e 10 allergen from olive pollen ( Olea europaea )

The Ole e 10 allergen has been purified from of olive pollen protein extract, obtained by homogenization of 3 g of pollen in 50 mM ammonium bicarbonate pH 8.0 for 3 hours at room temperature. Said extract was applied to a Sephadex G-75 column. The fractions that contained the protein were applied to a column of penetrability in Sephadex G-50 and finally at a C-18 reverse phase in HPLC with a gradient of acetonitrile (0-60%) in 0.1% TFA.

The invention covers the use of recombinant Ole e 10 in diagnosis, "in vivo" by skin tests or "in vitro" by immunodetection techniques (ELISA, RAST), allowing to specify which allergens are responsible for the clinical symptomatology of an individual and reduce the number of molecules necessary for your immunotherapy. Thus, a personalized immunotherapy is originated.

<110> Complutense University of Madrid

         \ vskip0.400000 \ baselineskip
      

<120>. Production, insulation system and purification of the Ole e 10 allergen.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<160> 2

         \ vskip0.400000 \ baselineskip
      

<210> 1

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Olea europaea

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> CDS

         \ vskip0.400000 \ baselineskip
      

<222> (1) ... (369)

         \ vskip0.400000 \ baselineskip
      

<300>

         \ vskip0.400000 \ baselineskip
      

<308> AY082335

         \ vskip0.400000 \ baselineskip
      

<309> 2002 03 19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

3

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 123

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Olea europaea

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> sig_peptide

         \ vskip0.400000 \ baselineskip
      

<222> FROM 1 TO 21

         \ vskip0.400000 \ baselineskip
      

<300>

         \ vskip0.400000 \ baselineskip
      

<308> AY082335

         \ vskip0.400000 \ baselineskip
      

<309> 2002 03 19

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

4

Claims (19)

1. Recombinant DNA molecules characterized by SEQ ID NO: 1 that encode a polypeptide with allergenic activity and that possess at least one common allergenic epitope with the Ole e 10 allergen of Olea europaea . 2. Recombinant DNA molecules, or modifications thereof, according to claim 1, which encode polypeptides with at least one epitope of Ole e 10 in its structure, attached to an additional polypeptide, or chemical modified or enzymatically. 3. Recombinant DNA molecules characterized by hybridizing under strongly restrictive conditions with the sequence described in SEQ ID NO: 1. 4. Recombinant DNA molecules characterized by presenting at least 60% identity with that described in SEQ ID NO: 1, containing a nucleotide sequence encoding a polypeptide with identical or reduced allergenic activity (hypoallergenic) with respect to Ole e 10 of European Olea . 5. Recombinant polypeptide corresponding to the Ole e 10 allergen of Olea europaea , or fragments of said polypeptide, according to claims 1 and 2, characterized by the sequence given in SEQ ID NO: 2 presenting the antigenicity of at least one Ole epitope e 10. 6. Recombinant allergen polypeptides Ole e 10 counterpart in other related species and not phylogenetically related, or fragments of said polypeptides according to claims 3 and 4 presenting the antigenicity of at least one epitope of Ole e 10. 7. Polypeptides that have a sequence of amino acids according to claims 5 and 6, and having a attenuated allergenicity. 8. Polypeptides or fragments of said Chenopodiaceous pollen polypeptides with at least one Ole e 10 epitope, according to previous claims, that recombinants bound to an additional polypeptide are produced. 9. Pollen polypeptides of Olea europaea , according to claims 5,6,7 and 8, which are chemically or enzymatically modified. 10. A eukaryotic expression vector, and preferably pPICZαA, containing the sequences characterized by SEQ ID NO: 1 according to claims 1,2,3 and 4 whose expression results in the production of the polypeptides characterized by SEQ ID NO: 2 according to claims 5,6,7,8 and 9. 11. A eukaryotic host organism, preferably Pichia pastoris yeast, transformed with the construction formed according to claim 10. 12. A method for producing the recombinant forms corresponding to the polypeptide sequences described in claims 5,6,7,8 and 9, in the eukaryotic system of yeast Pichia pastoris , and preferably of strain KM71, grown in rich medium, characterized by an ion exchange chromatography in DEAE-cellulose, one of penetrability in Sephadex G-50 and one in a column one of penetrability in Sephadex G-50 and one in a C-18 reverse phase HPLC column. 13. A method for producing the recombinant forms corresponding to the polypeptide sequences mentioned in claims 5,6,7,8 and 9, in the eukaryotic system of yeast Pichia pastoris and preferably of strain KM71 grown in minimal medium, characterized by a DEAE-cellulose exchange chromatography, one of penetrability in Sephadex G-50 and one in a C-18 reverse phase HPLC column. 14. A method for the isolation of Ole e 10 allergen from Olea europaea pollen using a Sephadex G-75 penetrability chromatography, a Sephadex G-50 penetrability chromatography and a C-18 nucleosyl reverse phase HPLC column . 15. Use of the peptides of the claims 5-9 for the preparation of Pharmacological compounds 16. Use of the molecules according to claims 1-9 in the "in vitro" diagnosis of allergy. 17. Application of recombinant molecules of claims 1-9 for vaccine design intended for allergy immunotherapy. 18. Application of recombinant molecules of claims 1-4 for producing peptides, according to claims 5-9, containing at least an allergenic T epitope, capable of acting as vaccines. 19. Application of recombinant molecules of claims 1-4 in the production of recombinant hypoallergenic isoforms that have decreased or nullified its binding to IgE for the treatment of allergies.