Purification, crystallization, X-ray diffraction analysis and phasing of a Fab fragment of monoclonal neuroantibody alphaD11 against nerve growth factor.
The rat monoclonal neuroantibody alphaD11 is a potent antagonist that prevents the binding of nerve growth factor (NGF) to its tyrosine kinase A receptor (TrkA) in a variety of systems, most notably in two in vivo systems linked to crucial pathological states, such as Alzheimer's disease and HIV infection. To provide further insights into the mechanism of action of this potentially therapeutic monoclonal antibody, structural studies of the antigen-binding fragment (Fab) of alphaD11 were performed. alphaD11 IgG2a immunoglobulin was obtained from hybridomas by in vitro tissue culture. The alphaD11 Fab crystallizes in two crystal forms. Form I belongs to space group P1, with unit-cell parameters a = 42.7, b = 50.6, c = 102.7 A, alpha = 82.0, beta = 89.1, gamma = 86.0 degrees. With two molecules in the asymmetric unit, V(M) is 2.3 A(3) Da(-1) and the solvent content is 46%. A complete data set has been collected at 2.7 A resolution on beamline XRD-1 (ELETTRA, Trieste, Italy). Form II belongs to space group C2, with unit-cell parameters a = 114.8, b = 69.4, c = 64.10 A, beta = 117.0 degrees. With one molecule in the asymmetric unit, V(M) is 2.4 A(3) Da(-1) and the solvent content is 48%. A complete data set has been collected at 1.7 A resolution on beamline ID14-1 (ESRF, Grenoble, France). Phasing was successfully performed by Patterson search techniques and refinement of the structures is currently under way. Crystal forms I and II display a close-packing pattern.
Covaceuszach
Sonia
S
LayLineGenomics SpA, Via di Castel Romano 100, I-00128 Roma, Italy. covaceu@sissa.it
Cassetta
Alberto
A
Cattaneo
Antonino
A
Lamba
Doriano
D
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D003460:Crystallization; D018360:Crystallography, X-Ray; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D008958:Models, Molecular; D020932:Nerve Growth Factor / Q000276:immunology*; D011487:Protein Conformation; D051381:Rats
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D020932:Nerve Growth Factor