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R. Rodríguez, Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain. Fax: +34 913944159, E-mail: email@example.com
The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiaeα-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.
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The incidence of type-1 allergies has increased over the last few years in developed areas, affecting as much as 20% of the population . Thus, one of the main focuses of research into allergy must be improvement of diagnostic and treatment methods. The use of well-standardized allergenic molecules in clinical tests and therapy, rather than extracts from a biological allergenic source, which usually are poorly characterized and contain undesirable components that can cause side-effects, has been recommended . Tree pollens are a major source of airborne antigens causing IgE-mediated type-1 allergic symptoms, and, of these, olive pollen has high clinical relevance in the Mediterranean area, especially Spain and Italy [3,4]. Ole e 1, the major allergen from this pollen, has been well characterized in terms of its structural (sequence, glycosylation degree, pI, etc.) and immunological features [5,6]. Along with other major and minor allergens from different sources, it exhibits a high degree of polymorphism, which makes a detailed study of its allergenic and antigenic epitopes difficult and prevents analysis of its three-dimensional structure.
Ole e 1 has previously been cloned and expressed as a fusion protein in Escherichia coli cells using the pGEX-2T vector . Although the response of the recombinant protein to IgG and IgE antibodies was comparable with that of the allergen isolated from the pollen, the expression system produced a low yield of soluble Ole e 1 after the purification process , most of the allergen being present as high-molecular-mass aggregates.
The methylotrophic yeast Pichia pastoris has been used to express a variety of biologically active proteins in extremely high yields [8–10]. Among them, two allergens, Cyn d 1 from Bermuda grass  and Bla g 4 from German cockroach , have been produced in this system. Secretion of heterologous proteins has been achieved using either their own signal peptide [13,14] or another signal peptide that is efficiently recognized by the yeast as the leader sequence of the Saccharomyces cerevisiaeα-mating factor  and the signal sequence of the Pichia acid phosphatase .
In this work we have succeeded in producing Ole e 1 in the heterologous system of Pichia pastoris yeast cells as a correctly folded efficiently processed secretion protein, allowing purification of a homogeneous soluble form of this allergen.
Materials and methods
Strains and plasmids
P. pastoris GS115 his4 strain (Invitrogen Corp.) was used as host for transformations. The E. coli strains used for DNA manipulations, DH5αF′ and TG1, were used as hosts for plasmids pUC18 (SmaI/bacterial alkaline phosphatase treated; Pharmacia Biotech) and pPIC9 (Invitrogen Corp.).
Construction of Ole e 1 expression vectors
The coding region of the Ole e 1 gene (clone OLE3c ) was amplified by PCR with DynaZyme™ DNA polymerase (Finnzymes Oy) using the plasmid pGEX-2T/Olee1 as template and two non-degenerate primers, a sense primer 5′-CGTCTCGAGAAAAGAGAGGATGTTCCGCA-3′ and an antisense primer 5′-GCGAATTCTCACATGTTGGGCGGGTA-3′, which, respectively, hybridized with the 5′ and 3′ ends of the protein-encoding region. The sense primer allows fusion of the protein-encoding region in-frame with the sequence coding for the preprosequence of the α-mating factor, present in plasmid pPIC9, and includes a XhoI restriction site (underlined). The antisense primer contains a stop codon and an EcoRI restriction site (underlined).
The DNA segment purified was treated with T4 DNA polymerase before phosphorylation treatment with T4 polynucleotide kinase . This fragment was ligated into a dephosphorylated SmaI-digested pUC18 plasmid vector as described . This construct was used to transform DH5αF′E. coli cells, and the DNA from several clones was sequenced as described by Sanger et al. , confirming the in-frame arrangement of the leader sequence and Ole e 1, as well as the absence of any change from the starting sequence. The DNA fragment was again digested with XhoI–EcoRI restriction enzymes and subcloned into the same sites of plasmid pPIC9 rendering pPIC9/Olee1.
Recombinant DNA methods other than those reported above were carried out by standard procedures .
Transformation of P. pastoris GS115 and expression of recombinant Ole e 1 (rOle e 1)
pPIC9/Olee1 (2 µg) was linearized with BglII restriction enzyme, and the purified larger fragment was used to transform GS115 cells by a replacement-type DNA integration using lithium acetate treatment as described . Transformed cells were incubated on minimal dextrose (MD) plates at 30 °C for 4–6 days until colonies appeared. Screening for gene replacement of the construct by homologous recombination at the AOX1 locus, rendering a (His+ Muts) phenotype, was performed by patching the His+ colonies in replica-plating on minimal dextrose vs. minimal methanol plates.
For the production of rOle e 1, selected (His+ Muts) transformed strains were cultured for 48 h at 30 °C in 50 mL buffered glycerol complex medium. Cells were then collected by centrifugation and resuspended in one-fifth of the original volume of buffered methanol complex medium for induction of the AOX1 promoter. This culture was maintained for 4 days and supplemented daily with 5 mL of methanol per litre of culture. The culture medium of GS115-induced cells was cleared of yeast cells by centrifugation at 3000 g at 4 °C. The production of rOle e 1 in the supernatant of the culture medium was analysed by SDS/PAGE by taking samples at different times (0, 24, 48, 96 h).
Large-scale production of rOle e 1 was performed under similar conditions using the colony that rendered the best yield in the small-scale experiments.
Production and purification of rOle e 1
The extracellular medium obtained after the induction was used, after dialysis against 20 mm ammonium bicarbonate, pH 8.0, as starting material to purify rOle e 1. Anion-exchange chromatography on a DEAE-cellulose column was used to fractionate the sample under a gradient (0.02–0.5 m) of ammonium bicarbonate. Fractions containing rOle e 1, as judged by SDS/PAGE, were lyophilized and chromatographed on a Sephadex G-75 size-exclusion column in 0.2 m ammonium bicarbonate. Fractions containing pure rOle e 1 were lyophilized.
Natural Ole e 1 (nOle e 1) was purified from olive pollen extract as described .
Analytical methods, protein digestion and amino-acid sequence
A reverse-phase HPLC on a Nucleosil C-18 column with an acetonitrile gradient (0–60%) in 0.1% trifluoroacetic acid was used to determine the homogeneity of these allergens. The eluate was continuously monitored at both 214 and 280 nm.
Protein concentration of purified samples (1–2 nmol) was determined by amino-acid analysis after hydrolysis with 5.7 m HCl at 105 °C for 24 h, in sealed tubes under vacuum. Hydrolysed samples were analysed on a 6300 amino acid analyser (Beckman Instruments).
Carboxyamidomethylated rOle e 1 (20 nmol) was digested with sequence-grade trypsin as described . Peptides were purified by reverse-phase HPLC on a Nucleosil C-18 column with an acetonitrile gradient in 0.1% trifluoroacetic acid. N-Terminal Edman degradation of the peptides was performed automatically using a 477A sequencer (Applied Biosystems-PE Corp.) .
Samples were mixed with a matrix solution comprised of saturated α-cyano-4-hydroxycinnamic acid in 30% aqueous acetonitrile and 0.1% trifluoroacetic acid. Samples were measured on a Bruker Reflex II matrix-assisted laser-desorption ionization time-of-flight mass spectrometer equipped with an ion source with visualization optics and a nitrogen laser (337 nm). Mass spectra were recorded in linear positive mode at 28.5 kV acceleration voltage and 1.6 kV in the linear detector accumulating 100 spectra of single laser shots under threshold irradiation. The equipment was externally calibrated employing singly, doubly and triply charged signals from either cytochrome c (12 360 Da) or bovine serum albumin (66 430 Da).
CD and fluorescence analyses
CD spectra were obtained on a Jasco J-715 spectropolarimeter as described  with minor modifications. The protein concentration was in the 0.20–0.25 mg·mL−1 range for the far-UV and 1 mg·mL−1 for the near-UV spectra. Mean residue mass ellipticities were calculated based on 111 as the average molecular mass/residue, obtained from the amino acid composition, and expressed in terms of θ (degree·cm2·dmol−1). Fluorescence emission spectra were obtained on an SLM Aminco 8000 spectrofluorimeter at 25 °C and in 0.2-cm optical-path cells, using 4-nm slits for both excitation and emission beams. The sample concentration was 0.25 mg·mL−1 in 20 mm ammonium bicarbonate, pH 8.0.
Sera and antibodies
Sera from hypersensitive individuals, which exhibited a positive reaction to Ole e 1, were selected from a population with a clinical history and a positive skin-prick test to olive pollen. Polyclonal serum against nOle e 1 was prepared by immunizing a New Zealand White rabbit over a 6-week period by weekly injection of the protein (100 µg) in complete Freund’s adjuvant. OL1, OL2 and OL3 monoclonal antibodies were obtained as described in  by Dr Lombardero (ALK-Abelló, Madrid, Spain), and OL4 monoclonal antibody was prepared as described in  by Dr Lahoz (Fundación Jiménez Díaz, Madrid, Spain).
Electrophoresis and immunological characterization
SDS/PAGE was performed by the method of Laemmli  in 15% polyacrylamide gels. Proteins were stained with Coomassie blue or electrophoretically transferred to nitrocellulose membranes. Immunodetection was achieved as described  by using four monoclonal antibodies, a pool of sera from patients allergic to olive pollen (diluted 1 : 10), or a rabbit polyclonal antiserum raised against nOle e 1 (diluted 1 : 5000). The signal was developed by the ECL-Western-blotting reagent (Amersham Corp.) .
ELISA inhibition assays were performed as described . After being coated with 100 µL antigen (1 µg·mL−1), the plates were incubated with the pool of sera (diluted 1 : 10), previously incubated with different concentrations of nOle e 1 or rOle e 1 (0.01–100 ng) as inhibitors; this was followed by incubation with mouse anti-(human IgE) and horseradish peroxidase-labelled goat anti-(mouse IgG).
Carbohydrate detection and deglycosylation treatment
Carbohydrate detection of protein transferred to nitrocellulose membranes was performed as previously described  by using a biotinylated concanavalin A solution. The staining was developed by horseradish peroxidase reaction with 0.05% diaminobenzidine/0.03% of 30% H2O2 in Tris/NaCl reaction buffer.
Enzymatic deglycosylation was performed with PNGase F endoglycosidase (Boehringer-Mannheim) as described .
Expression of Ole e 1
The coding region of Ole e 1 was amplified by PCR using the plasmid pGEX-2T/Olee1 as a template and the two primers described in the Materials and Methods section. After cloning of cDNA encoding Ole e 1 allergen (clone OLE3c ) in the pPIC9 expression plasmid in-frame with the rest of the leader sequence, the vector carrying the Ole e 1 cDNA inserted downstream of the AOX1 promoter (pPIC9/Olee1) was used for transformation of P. pastoris GS115 cells obtaining the efficient extracellular secretion of rOle e 1. The time course of the expression was examined in the secreted medium by SDS/PAGE (Fig. 1A). A major band of apparent molecular mass 20.5 kDa can be observed, the highest level of production being at 96 h. To confirm that the expressed protein was Ole e 1, electrophoresed samples were transferred to nitrocellulose membranes and immunostained with an Ole e 1-specific polyclonal antibody, giving a positive reaction (data not shown). After selection of the conditions of growth, one of the transformed colonies, which produced 60 mg allergen per litre of cell culture, was selected for isolation of the protein.
Purification of rOle e 1 and molecular properties
A two-step procedure consisting of anion-exchange and size-exclusion chromatography was used to purify rOle e 1. The extracellular medium of the culture was exhaustively dialysed to remove small contaminants. Afterwards, the medium was fractionated on a DEAE-cellulose column and the eluted material analysed by SDS/PAGE (Fig. 1B). Fractions containing rOle e 1 were then loaded on a Sephadex G-75 column and the elution profile examined as the previous chromatography (Fig. 1B). The final yield of the purified protein was 35 mg of allergen per litre of cell culture.
rOle e 1 exhibited an apparent molecular mass of 20.5 kDa, which was slightly higher than that of the glycosylated form of the natural allergen (20.0 kDa) and significantly higher than that obtained for the unglycosylated variant (18.5 kDa) . To try to explain this difference, the presence of carbohydrates in rOle e 1 was analysed by reaction of the protein transferred to a nitrocellulose membrane with biotinylated concanavalin A. As Fig. 2A shows, rOle e 1 gave a positive response in this treatment. Deglycosylation of both natural and recombinant allergens with the PNGase F endoglycosidase was carried out to confirm that the molecular mass difference was due exclusively to the carbohydrate moiety. SDS/PAGE analysis of the glycosidase-treated nOle e 1 and rOle e 1 proteins showed identical mobilities to the unglycosylated forms (Fig. 2B). This result was confirmed by MS analysis of rOle e 1 that had been treated or not with PNGase F (Fig. 3). The former gave a single homogeneous peak, whereas the untreated sample showed four different peaks ranging between 18 226 and 18 702 Da. The difference between the molecular mass of each molecular species and its closest neighbours is around 160 Da, which corresponds to one mannose residue. This would agree with the existence of several forms of rOle e 1 with different degrees of glycosylation (18 226, 18 381, 18 534 and 18 702 Da could correspond to the polypeptide chain plus, respectively, Man9GlcNAc2, Man10GlcNAc2, Man11GlcNAc2 and Man12GlcNAc2).
The recombinant allergen was characterized by different analytical methods to evaluate its purity as well as its similarity to the natural allergen obtained from the pollen. The homogeneity of rOle e 1, which corresponds to one of the polypeptide isoforms of nOle e 1 (clone OLE3c), can be observed in the elution profile of the expressed protein in analytical reverse-phase HPLC (Fig. 4) when it is compared with that of the polymorphic nOle e 1. The amino acid composition of rOle e 1 (data not shown) agrees with that obtained from the deduced amino acid sequence of clone 3c. The N-terminus and five peptides of rOle e 1 were sequenced by Edman degradation. The amino acid sequences determined (EDVPQPP, LQCKDKENGDVT, AEGLYSMLVER, TVNPLGFFK, EALPK) were in agreement with those expected from positions 1–7, 41–52, 61–71, 116–124, and 126–130 of the polypeptide chain of Ole e 1 (clone OLE3c), respectively.
Accurate evidence for the correct folding of rOle e 1 was obtained by spectroscopic studies comparing it with the natural allergen. Far- and near-UV CD spectra of both proteins were obtained (Fig. 5A,B). No significant differences between them were found, not only in terms of the shape of the spectra but also with respect to the molar ellipticity values. Fluorescence spectroscopy data for nOle e 1 and rOle e 1 were also analysed; they were found to exhibit overlapping curves (Fig. 5C). These data indicate that the expressed allergen is correctly folded at the levels of secondary and tertiary structure.
A comparison of the IgG- and IgE-binding activities of rOle e 1 and nOle e 1 was also carried out. Four monoclonal antibodies, OL1, OL2, OL3 and OL4, raised against nOle e 1 were used for immunostaining of Western blotted proteins (Fig. 6A). All exhibited strong reactivity to nOle e 1 and rOle e 1 electrophoresed under non-reducing conditions. However, the last three did not recognize denatured Ole e 1. These results indicate that rOle e 1 possesses the antigenic determinants, and therefore the native conformation, required for binding to these antibodies. Immunoblotting analysis using a polyclonal antibody raised against nOle e 1 exhibited similar reactivity to both the natural and recombinant forms (Fig. 6B). In addition, a serum pool of five olive-allergic patients sensitive to Ole e 1 showed comparable IgE binding to the two proteins (Fig. 6C).
IgE binding to rOle e 1 and nOle e 1 was quantitatively evaluated in ELISA inhibition experiments, in which each one was alternately coated to the wells. The two proteins exhibited the same extent of inhibition of binding to IgE antibodies (Fig. 7), indicating that they are equivalent at the immunological level.
Two of the main objectives in allergy research is to characterize the allergenic molecules under investigation and produce them in high enough yield to use them in diagnosis and treatment of the disease. This report describes the production, purification and characterization of a soluble and correctly folded recombinant form of Ole e 1, the most prevalent allergen from olive tree pollen, using the methylotrophic yeast P. pastoris.
Ole e 1 presents a frequency in the reactivity of the patients allergic to olive pollen of more than 60%, reaching 80% depending on the geographical area in which the sensitization occurs. Its most relevant structural features include three disulfide bonds, one glycosylation site at Asn111 and a high degree of polymorphism (nOle e 1 is actually a mixture of polypeptides with different glycosylation patterns). It had previously been obtained as a recombinant allergen using an E. coli system with the pGEX-2T vector and synthesized as a fusion protein (GST-Ole e 1) . Disulfide-bonded proteins such as Ole e 1 usually cannot be produced successfully by expression in bacteria because the cytoplasm is a reducing environment . Therefore, most proteins with these characteristics have to be refolded from their insoluble forms produced in inclusion bodies. As a result, one of the main limitations of this method of expression is the low yield of the recombinant protein. In the approach we used for the expression of Ole e 1 in E. coli, the amount of correctly folded protein was as low as 200 µg per litre, although immunological properties were well preserved .
P. pastoris has become a potentially attractive host for the expression of foreign genes and has been successfully utilized as an alternative expression system because the proteins are usually obtained as secreted forms in large amounts and with the correct disulfide bonds . In addition, the carbohydrate cores incorporated into the N-glycosylation sites are sometimes similar to those of the natural glycoproteins . Moreover, one of the main advantages in producing heterologous proteins as secreted products in P. pastoris is the easy isolation of the recombinant molecule from the medium in which it is produced, because it is completely soluble and has a high degree of initial purity because the level of endogenous secreted proteins is very low. Ole e 1 was therefore expressed in this system at high yield over the course of 96 h, at which time the recombinant protein is proteolytically stable. It is fully active in terms of its antigenic and allergenic features, as determined using IgE and monoclonal IgG antibodies, and can be easily purified to homogeneity using a two-step procedure with conventional chromatography columns. Another advantage of this protocol is the attainment of a native conformation. Many secreted proteins are thiol rich, their disulfide bonds contributing to achieving and maintaining the correct tertiary structure. The secretory pathway and the extracellular medium of P. pastoris are oxidizing environments which can support the formation of these disulfide bonds. Maintenance of the native conformation in rOle e 1 was assessed by CD and fluorescence analyses, which provide information about the secondary and tertiary structures of the allergen, as well as by the study of its interaction with IgG monoclonal antibodies that bind to conformational epitopes.
The preparation of rOle e 1 appeared to be homogeneous as assessed by SDS/PAGE, HPLC and amino acid sequencing. The microheterogeneity detected in the mass spectra of the expressed allergen was not due to differences in the polypeptide structure but to the glycosidic moiety. rOle e 1 is, in fact, a unique protein species with different degrees of glycosylation, as it renders, after deglycosylation with PNGase F, a band at the same position as that of unglycosylated nOle e 1 in SDS/PAGE, as well as one unique homogeneous peak at the molecular mass of the free polypeptide chain in MS. The extensive polymorphism of the natural allergen, which makes it unsuitable for epitope analysis and three-dimensional studies, is avoided by the recombinant production.
Production of secreted proteins in P. pastoris involves post-translational modification of the polypeptide chain by the addition of N-asparagine-linked oligosaccharides, which exhibit a pattern of different Man8–13(GlcNAc)2 isomers [27,28]. MS analysis of rOle e 1 showed four main isoforms, the molecular masses of which correlated with the presence of 9–12 Man residues attached to the core of two GlcNAc moieties of the asparagine-linked oligosaccharides of glycoproteins.
To complete the characterization of the protein and to confirm the immunological integrity of the purified rOle e 1, the reactivities of Ole e 1-specific antibodies and allergic sera were analysed. On immunoblotting, comparable reactivities were observed for rOle e 1 and nOle e 1 allergens using four monoclonal antibodies obtained against the natural allergen and belonging to different families. This result is very significant if we take into account that three of them only recognize the protein in its native conformation. ELISA inhibition analysis, which is highly sensitive, revealed very similar IgE-binding activity for the recombinant and natural proteins. The small differences observed could be due to either the polymorphic nature of nOle e 1 (some heterogeneous positions could be involved in the specific recognition by the IgE from the sera) or the N-glycosylated forms of rOle e 1, which are different from those of nOle e 1. These results indicate that the antigenic/allergenic epitopes of the natural allergen were obtained in the expressed protein and were well preserved during the purification steps. Therefore, the protein produced in this way may be used for clinical purposes. Moreover, the production of large amounts of homogeneous soluble correctly folded rOle e 1 is a requirement for study of the three-dimensional structure. It will be particularly useful for NMR analysis  because the production of the allergen with 2H, 13C or 15N would facilitate the recovery and processing of the data. As has happened for many proteins, solving the tertiary structure of Ole e 1 could shed light on its biological function, which is so far not known.
Finally, although a few allergens have already been expressed by the yeast system [11,12], the success of this attempt forecasts good results for proteins that cannot be satisfactorily produced in bacterial hosts.
This work was supported by Grant PM95–0074 from the Dirección General de Investigación Científica y Técnica (Spain). S. H., E. G. and A. M.-R. are recipients of predoctoral fellowships from the Ministerio de Educación y Ciencia (Spain). We thank Drs M. Lombardero and C. Lahoz for kindly donating the monoclonal antibodies.