Expression, crystallization, and preliminary X-ray analysis of a sialic acid-binding fragment of sialoadhesin in the presence and absence of ligand.
Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycoconjugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD1) and selenomethionyl (Se-SnD1) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnD1 and Se-SnD1 have been crystallized in the absence of ligand, and SnD1 has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnD1 and Se-SnD1 were isomorphous, of space group P3(1)21 or P3(2)21, with unit cell dimensions a = b 38.9 A,c = 152.6 A, alpha = beta = 90 degrees, gamma = 120 degrees, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P2(1)2(1)2(1), with unit cell dimensions a = 40.9 A, b = 97.6 A,c = 101.6 A, alpha = beta = gamma = 90 degrees.
May
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AP
Laboratory of Molecular Biophysics, University of Oxford, United Kingdom.
Robinson
R C
RC
Aplin
R T
RT
Bradfield
P
P
Crocker
P R
PR
Jones
E Y
EY
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018360:Crystallography, X-Ray; D008024:Ligands; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011971:Receptors, Immunologic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D063267:Sialic Acid Binding Ig-like Lectin 1
D008024:Ligands; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011971:Receptors, Immunologic; D063267:Sialic Acid Binding Ig-like Lectin 1; D019158:N-Acetylneuraminic Acid