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Purification, crystallization and preliminary crystallographic characterization of the alpha 2,6-sialyltransferase from Photobacterium sp. JT-ISH-224.

Okino N, Kakuta Y, Kajiwara H, Ichikawa M, Takakura Y, Ito M, Yamamoto T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

Bottom Line: Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids.The newly cloned alpha2,6-sialyltransferase from Photobacterium sp.The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 90.29, c = 204.33 A.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned alpha2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase. The putative mature form of JT-ISH-224 alpha2,6-sialyltransferase was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 90.29, c = 204.33 A. X-ray diffraction data were collected to 2.5 A resolution.

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A crystal of ISH224 α2,6-STase obtained using the hanging-drop vapour-diffusion method. The approximate dimensions of the crystal are 0.3 × 0.3 × 0.3 mm.
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fig1: A crystal of ISH224 α2,6-STase obtained using the hanging-drop vapour-diffusion method. The approximate dimensions of the crystal are 0.3 × 0.3 × 0.3 mm.

Mentions: Initial crystallization trials were carried out by the hanging-drop vapour-diffusion method in a 24-well plate at 293 K using the Crystal Screen, Crystal Screen 2 and Index Screen kits (Hampton Research). Each drop was prepared by mixing 1 µl protein solution (10 mg ml−1) containing 10 mM cytidine monophosphate (CMP) and 10 mM lactose and an equal volume of reservoir solution. After 5 d, a small crystal of α2,6-­STase was observed using a reservoir condition consisting of 0.2 M lithium sulfate monohydrate, 0.1 M Tris–HCl pH 8.5 and 30%(w/v) PEG 4000 (Fig. 1 ▶). This result was found to be reproducible. Under these conditions, crystals grew within 10 d to a sufficient size for data collection. Prior to data collection, the crystal was transferred stepwise into cryoprotectant solution consisting of the crystallization condition containing 10% glycerol and flashed-cooled in a nitrogen-gas stream at 100 K. X-ray diffraction data were collected using synchrotron radiation (1.000 Å wavelength) with a Jupiter 210 detector (Rigaku/MSC Corporation) at beamline BL38B1 of SPring-8 (Hyogo, Japan). The oscillation angle was 1.0° and the exposure time was 10.0 s per frame. A total of 180 diffraction images were collected at a camera distance of 150 mm and were processed using HKL-2000 (Otwinowski, 1993 ▶).


Purification, crystallization and preliminary crystallographic characterization of the alpha 2,6-sialyltransferase from Photobacterium sp. JT-ISH-224.

Okino N, Kakuta Y, Kajiwara H, Ichikawa M, Takakura Y, Ito M, Yamamoto T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2007)

A crystal of ISH224 α2,6-STase obtained using the hanging-drop vapour-diffusion method. The approximate dimensions of the crystal are 0.3 × 0.3 × 0.3 mm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335162&req=5

fig1: A crystal of ISH224 α2,6-STase obtained using the hanging-drop vapour-diffusion method. The approximate dimensions of the crystal are 0.3 × 0.3 × 0.3 mm.
Mentions: Initial crystallization trials were carried out by the hanging-drop vapour-diffusion method in a 24-well plate at 293 K using the Crystal Screen, Crystal Screen 2 and Index Screen kits (Hampton Research). Each drop was prepared by mixing 1 µl protein solution (10 mg ml−1) containing 10 mM cytidine monophosphate (CMP) and 10 mM lactose and an equal volume of reservoir solution. After 5 d, a small crystal of α2,6-­STase was observed using a reservoir condition consisting of 0.2 M lithium sulfate monohydrate, 0.1 M Tris–HCl pH 8.5 and 30%(w/v) PEG 4000 (Fig. 1 ▶). This result was found to be reproducible. Under these conditions, crystals grew within 10 d to a sufficient size for data collection. Prior to data collection, the crystal was transferred stepwise into cryoprotectant solution consisting of the crystallization condition containing 10% glycerol and flashed-cooled in a nitrogen-gas stream at 100 K. X-ray diffraction data were collected using synchrotron radiation (1.000 Å wavelength) with a Jupiter 210 detector (Rigaku/MSC Corporation) at beamline BL38B1 of SPring-8 (Hyogo, Japan). The oscillation angle was 1.0° and the exposure time was 10.0 s per frame. A total of 180 diffraction images were collected at a camera distance of 150 mm and were processed using HKL-2000 (Otwinowski, 1993 ▶).

Bottom Line: Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids.The newly cloned alpha2,6-sialyltransferase from Photobacterium sp.The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 90.29, c = 204.33 A.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka, Japan.

ABSTRACT
Sialyltransferases transfer sialic acid from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the nonreducing termini of the oligosaccharyl structures of various glycoproteins and glycolipids. The newly cloned alpha2,6-sialyltransferase from Photobacterium sp. JT-ISH-224 (from the Vibrionaceae family) is composed of two domains: an unknown N-terminal domain and a catalytic C-terminal domain which shares significant homology with the Pasteurella multocida multifunctional sialyltransferase. The putative mature form of JT-ISH-224 alpha2,6-sialyltransferase was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method at 293 K. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 90.29, c = 204.33 A. X-ray diffraction data were collected to 2.5 A resolution.

Show MeSH
Related in: MedlinePlus