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Acta Crystallogr Sect F Struct Biol Cryst Commun. Feb 1, 2005; 61(Pt 2): 186–188.
Published online Jan 8, 2005. doi:  10.1107/S1744309104033822
PMCID: PMC1952250

Crystallization and preliminary structure determination of the plant food allergen Pru av 2

Abstract

Thaumatin-like proteins (TLPs) have mostly been investigated in the context of their function as pathogenesis-related proteins and only in recent years have some of them been classified as allergens. Here, the purification and crystallization of the first allergenic TLP, Pru av 2, a 23.3 kDa protein isolated from ripe cherries, is reported. The crystals diffracted to 2.1 Å resolution at a rotating-anode generator and were found to belong to space group P21, with unit-cell parameters a = 44.48, b = 41.04, c = 59.16 Å, β = 106.61° and one molecule per asymmetric unit. In order to obtain high-resolution data, an annealing protocol was applied that improved the resolution limit from 1.6 to 1.3 Å at a synchrotron.

Keywords: food allergens, thaumatin-like proteins, cherries, crystal annealing

1. Introduction

Almost 4% of the human population suffer from food allergy. Interestingly, most plant food allergens belong to very few protein families and superfamilies (Breiteneder & Radauer, 2004 [triangle]). Of those, members of the pathogenesis-related proteins (PRs) have frequently been identified as allergens (Ebner et al., 2001 [triangle]; Hoffmann-Sommergruber, 2000 [triangle]). PRs are defined as proteins that are encoded by the plant genome and induced specifically in response to infections by fungi, bacteria or viruses or by adverse environmental conditions. PRs have been classified into 14 families by van Loon & van Strien (1999 [triangle]). The family 5 PRs share significant amino-acid sequence similarity with thaumatin, an intensely sweet-tasting protein from the fruit of the West African ketemfe plant (Thaumatococcus daniellii; Van der Wel & Loeve, 1972 [triangle]). Therefore, these proteins are called thaumatin-like proteins (TLPs). They have been isolated from a wide variety of plant species (Selitrennikoff, 2001 [triangle]). A number of allergenic TLPs from fruit and pollen have already been described (Breiteneder & Radauer, 2004 [triangle]). TLPs for which specific IgE reactivity has been described include apple Mal d 2 (Hsieh et al., 1995 [triangle]; Krebitz et al., 2003 [triangle]), cherry Pru av 2 (Inschlag et al., 1998 [triangle]), bell pepper Cap a 1 (Fuchs et al., 2002 [triangle]), the kiwi TLP Act c 2 (Gavrović-Jankulović et al., 2002 [triangle]) and the pollen TLPs Jun a 3 (Midoro-Horiuti et al., 2000 [triangle]) and Cup a 3 (Cortegano et al., 2004 [triangle]).

To date, the structures of four plant TLPs have been solved (Batalia et al., 1996 [triangle]; Koiwa et al., 1999 [triangle]; Min et al., 2004 [triangle]; Ogata et al., 1992 [triangle]), but nothing is known about their allergenic potential and IgE cross-reactivity within the TLP family in general. In order to close this gap in our knowledge, we have initiated structural and immunological investigations of TLP proteins. Here, we present the purification and crystallization of Pru av 2, a 23.3 kDa allergen (222 amino acids) which was isolated from sweet cherry. Pru av 2 is the first allergenic fruit TLP to be crystallized.

2. Protein purification

The TLP from sweet cherry was purified according to a previously described protocol (Menu-Bouaouiche et al., 2003 [triangle]), with some modifications. In brief, 1.5 kg ripe cherries (Prunus avium cv. Germersdorfer) grown in Northern Austria were squeezed with 300 ml ice-cold water through Miracloth and diluted in sodium formate buffer to a final concentration of 20 mM. The extract was adjusted to pH 3.8 with 100% formic acid and then centrifuged at 9000g for 30 min at 277 K. After filtration of the supernatant, the cherry protein extract was further diluted with distilled water to 1200 ml and loaded onto a column of 25 ml SP-Sepharose Fast Flow (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM sodium formate buffer pH 3.8 and 1 M NaCl. All chromatographic steps on the ÄKTA-Prime System (Amersham) were performed at 277 K. After loading the sample, the column was washed with 20 mM sodium formate buffer pH 3.8. Proteins were subsequently eluted using a 200 ml linear gradient of 0–200 mM NaCl in 50 mM Tris–HCl pH 7.8. 10 ml fractions were collected and analyzed by SDS–PAGE.

The Pru av 2 protein was detected in the flowthrough and was highly purified after the washing step. All other proteins were retained on the column. The Pru av 2 present in 600 ml of the washing step was precipitated by adding ammonium sulfate to a final concentration of 75%. After centrifugation, the precipitate was dissolved in 5 ml 20 mM sodium phosphate buffer pH 7.2 and desalted using Sephadex G-25 prepacked PD-10 columns (Amersham Biosciences, Uppsala, Sweden), resulting in a diluted volume of 17 ml. The fractions containing the purified Pru av 2 protein were pooled. Prior to crystallization experiments, the buffer was replaced by water and the protein was concentrated to 9 mg ml−1 using Ultrafree-MC filter units (Millipore). The protein concentration was determined by the BCA Protein Assay (Pierce Biotechnology, Perbio Science, Erembodegem, Belgium) using bovine serum albumin as standard.

3. Crystallization

Initial crystallization conditions for Pru av 2 were screened at 293 K using the Index Screen (Hampton Research Inc.) and the microbatch method with the ORYX crystallization robot (Douglas Instruments). Small crystals were obtained from a condition consisting of 25%(w/v) PEG 3350 and 0.1 M citric acid pH 3.5. The drops contained 0.5 µl protein and 0.5 µl precipitant solution. The precipitant concentration and the pH of the citric acid were optimized and the best crystals were obtained using solutions consisting of 26–30%(w/v) PEG 3350 and 0.1 M citric acid pH 3.35–3.5. Plate-like crystals formed within a week and grew to maximum dimensions of 0.55 × 0.15 × 0.1 mm (Fig. 1 [triangle]).

Figure 1
A representative crystal of Pru av 2 grown using the microbatch method at 293 K in 26%(w/v) PEG 3350, 0.1 M citric acid pH 3.35.

4. Data collection and processing

The first complete data set was collected using a system with a MAR 345 image-plate detector and a rotating-anode generator operating at 40 kV and 80 mA. Crystals were cooled using flash-cooling in liquid nitrogen without cryoprotectant. Data were collected at 100 K by the standard oscillation method using a crystal-to-detector distance of 80 mm, an oscillation range of 1.0° and an exposure time of 20 min per image. The crystal diffracted to 2.1 Å and no radiation damage was detected during data collection. The crystal belonged to space group P21, with unit-cell parameters a = 44.48, b = 41.04, c = 59.16 Å, β = 106.61°, and had a mosaicity of 0.75°. Assuming the presence of one molecule of Pru av 2 in the asymmetric unit, the Matthews coefficient (Matthews, 1968 [triangle]) is 2.1 Å3 Da−1 and the solvent content is 44.1%. Data processing and reduction were performed with DENZO and SCALEPACK (Otwinowski & Minor, 1997 [triangle]). Crystal parameters and diffraction data statistics are summarized in Table 1 [triangle].

Table 1
Data-collection and processing statistics

In order to obtain a high-resolution structure, data collection was carried out at beamline X11, EMBL/DESY Hamburg, Germany equipped with a MAR Research CCD detector. For data collection, the crystals were flash-cooled in liquid nitrogen after soaking in cryoprotectant solution containing 20%(w/v) glycerol. Under these conditions, the crystals diffracted to 1.6 Å. Repeated cycles of annealing (warming up the crystal by interrupting the cold stream, followed by recooling to 100 K in the gaseous nitrogen stream) resulted in the extension of the resolution limit to 1.3 Å, with a simultaneous increase of the mosaicity to 0.4°. Fig. 2 [triangle] shows a comparison of the diffraction pattern before and after the annealing procedure.

Figure 2
The diffraction pattern of a Pru av 2 crystal using synchrotron radiation at beamline X11, DESY, Hamburg (a) before annealing (the outer edge of the plate is at 1.6 Å), (b) after annealing (the outer edge of the plate is at 1.15 Å). ...

Database searches with the program GenTHREADER (Jones, 1999 [triangle]) revealed that thaumatin is the structure closest related to Pru av 2, exhibiting 41.5% sequence identity. The whole thaumatin structure (PDB code http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1thv) was used as a model for molecular replacement. A clear solution was obtained with MOLREP (Collaborative Computational Project, Number 4, 1994 [triangle]) using data from 18 to 3 Å, where the first peak in the translation function showed a relative height (TF/σ) of 11.6, compared with 5.3 for the second peak.

Model building and refinement is in progress and upon completion the Pru av 2 structure will form the basis for an analysis of allergenicity and cross-reactivity within the TLP family of allergens.

Acknowledgments

This work was supported by the Austrian Science Fund (FWF) projects F01802 and F01805. Support during data collection at DESY by the EMBL staff is gratefully acknowledged. Data collection was supported by a travel grant from the European Community (Access to Research Infrastructure Action of the Improving Human Potential Programme to the EMBL Hamburg Outstation, contract No. HPRI-CT-1999-00017).

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