Purification, crystallization and X-ray diffraction analysis of a recombinant Fab that recognizes a human blood-group antigen.
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).
Song
Shuh Chyung
SC
Department of Pathology and Laboratory of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Xie
Kefang
K
Czerwinski
Marcin
M
Spitalnik
Steven L
SL
Wedekind
Joseph E
JE
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
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D008951:MNSs Blood-Group System; D011994:Recombinant Proteins