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Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

Kezuka Y, Itagaki T, Satoh R, Teshima R, Nonaka T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2009)

Bottom Line: A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis.A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

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Crystals of rBWp16ΔN (a) and its SeMet derivative (b). The scale bars correspond to 0.1 mm. The crystals in (a) and (b) were obtained under conditions I and II, respectively.
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fig1: Crystals of rBWp16ΔN (a) and its SeMet derivative (b). The scale bars correspond to 0.1 mm. The crystals in (a) and (b) were obtained under conditions I and II, respectively.

Mentions: After trials of approximately 400 conditions in the initial screening, an rBWp16ΔN crystal was obtained under condition No. 40 of the Cryo I kit from Emerald BioSystems [40%(v/v) ethanol, 100 mM phosphate–citrate pH 4.2, 5%(w/v) PEG 1000]. However, we did not succeed in crystallizing rBWp16. Therefore, the deletion of the 12 N-­terminal residues was likely to affect crystallization. Crystallization conditions were optimized for rBWp16ΔN and the following two promising conditions were established. One is a slight modification of Cryo I condition No. 40 [36%(v/v) ethanol, 0.1 M phosphate–citrate pH 4.2, 1%(w/v) PEG 1000; condition I]. The other was 29%(v/v) 1-­propanol, 0.1 M phosphate–citrate pH 4.2, 1%(w/v) PEG 1000 (condition II). Plate-like crystals grew within 2 d and reached a maximum dimension of 0.2 mm (Fig. 1 ▶ a); dissolution occurred within several days under both conditions. A complete data set was collected to 1.72 Å resolution from a native crystal obtained under condition I. Table 1 ▶ gives a summary of the data-collection and data-processing statistics. The native crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 27.92, b = 58.54, c = 32.16 Å, β = 109.34°. The calculated Matthews coefficient of 1.87 Å3 Da−1 (Matthews, 1968 ▶), corresponding to a solvent content of 34%, indicated the presence of one monomer in the asymmetric unit.


Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

Kezuka Y, Itagaki T, Satoh R, Teshima R, Nonaka T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2009)

Crystals of rBWp16ΔN (a) and its SeMet derivative (b). The scale bars correspond to 0.1 mm. The crystals in (a) and (b) were obtained under conditions I and II, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802877&req=5

fig1: Crystals of rBWp16ΔN (a) and its SeMet derivative (b). The scale bars correspond to 0.1 mm. The crystals in (a) and (b) were obtained under conditions I and II, respectively.
Mentions: After trials of approximately 400 conditions in the initial screening, an rBWp16ΔN crystal was obtained under condition No. 40 of the Cryo I kit from Emerald BioSystems [40%(v/v) ethanol, 100 mM phosphate–citrate pH 4.2, 5%(w/v) PEG 1000]. However, we did not succeed in crystallizing rBWp16. Therefore, the deletion of the 12 N-­terminal residues was likely to affect crystallization. Crystallization conditions were optimized for rBWp16ΔN and the following two promising conditions were established. One is a slight modification of Cryo I condition No. 40 [36%(v/v) ethanol, 0.1 M phosphate–citrate pH 4.2, 1%(w/v) PEG 1000; condition I]. The other was 29%(v/v) 1-­propanol, 0.1 M phosphate–citrate pH 4.2, 1%(w/v) PEG 1000 (condition II). Plate-like crystals grew within 2 d and reached a maximum dimension of 0.2 mm (Fig. 1 ▶ a); dissolution occurred within several days under both conditions. A complete data set was collected to 1.72 Å resolution from a native crystal obtained under condition I. Table 1 ▶ gives a summary of the data-collection and data-processing statistics. The native crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 27.92, b = 58.54, c = 32.16 Å, β = 109.34°. The calculated Matthews coefficient of 1.87 Å3 Da−1 (Matthews, 1968 ▶), corresponding to a solvent content of 34%, indicated the presence of one monomer in the asymmetric unit.

Bottom Line: A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis.A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

Show MeSH
Related in: MedlinePlus