title: Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.
abstract: The NNA7 Fab fragment recognizes the human glycopeptide N blood-group antigen and has a high affinity for N-type glycophorin A (GPA). To provide insight into how antibodies recognize glycopeptide antigens, soluble Fab fragments were expressed in Escherichia coli, purified and crystallized using the hanging-drop vapor-diffusion method at 293 K. The best crystals were obtained from solutions of PEG monomethyl ether 5000 containing 4-8 mM yttrium chloride (YCl3). This rare-earth ion, which could be substituted with various lanthanides, changed the habit of crystals from multinucleated rods with a diffraction limit of 4.25 A resolution to a diamond-shaped morphology that grew as single crystals and diffracted X-rays to 1.75 A resolution. Data were collected that indicated that the crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 77.1, c = 118.1 A and one Fab fragment per asymmetric unit. A molecular-replacement solution has been obtained and 86% of the molecule was fitted by use of an automated refinement procedure (ARP).
authors.affiliation: Department of Pathology and Laboratory of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
authors.forename: Shuh Chyung
authors.initials: SC
authors.lastname: Song
authors.forename: Kefang
authors.initials: K
authors.lastname: Xie
authors.forename: Marcin
authors.initials: M
authors.lastname: Czerwinski
authors.forename: Steven L
authors.initials: SL
authors.lastname: Spitalnik
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
mesh_terms: D000936:Antigen-Antibody Complex / Q000737:chemistry; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology; D008951:MNSs Blood-Group System / Q000737:chemistry* / Q000276:immunology; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D008951:MNSs Blood-Group System; D011994:Recombinant Proteins