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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 deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.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).

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 deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.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).

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