Combined use of AFM and X-ray diffraction to analyze crystals of an engineered, domain-deleted antibody.
A genetically engineered humanized C(H)2-domain-deleted monoclonal antibody lacking any interchain-hinge disulfide bonds has been crystallized in the presence of detergent in a form suitable for X-ray diffraction analysis. The crystals were grown from 4 M formate along with Triton X-100 and had P2(1)2(1)2 space-group symmetry, with unit-cell parameters a = 83, b = 224, c = 167 A. The crystals diffract to beyond 2.8 A resolution. A disordered crystal form of larger size and more attractive habit was also grown from 4 M formate, but in the presence of the Anapoe series of detergents. Preliminary X-ray data, in conjunction with atomic force microscopy images, are consistent with asymmetric units consisting of two intact antibodies forming a circular dimeric ring. The crystallizing unit, which must contain a twofold axis, is a toroidal assembly of four antibodies (two dimeric rings). Competition between dimers and tetramers to enter the lattice, along with a unique kind of planar defect of packing, may be responsible for the unusually high defect density and the disorder of the X-ray diffraction pattern exhibited by the second crystal form. An approach to crystallizing proteins showing phase separation, particularly intact antibodies, that uses a preliminary detergent test set is described.
Larson
Steven B
SB
University of California, Irvine, Department of Molecular Biology and Biochemistry, Irvine, CA 92697-3900, USA.
Kuznetsov
Yu G
YG
Day
John
J
Zhou
Jiashu
J
Glaser
Scott
S
Braslawsky
Gary
G
McPherson
Alexander
A
D000912:Antibodies, Neoplasm / Q000737:chemistry*; D000951:Antigens, Neoplasm / Q000276:immunology; D003460:Crystallization; D018360:Crystallography, X-Ray; D003902:Detergents; D006023:Glycoproteins / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D017830:Octoxynol; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D003902:Detergents; D006023:Glycoproteins; D007128:Immunoglobulin Fragments; C047052:tumor-associated antigen 72; D017830:Octoxynol