Fab crystallization and preliminary X-ray analysis of NC-1, an anti-HIV-1 antibody that recognizes the six-helix bundle core of gp41.
NC-1 is a murine monoclonal antibody that specifically recognizes the six-helix bundle core of the human immunodeficiency virus type 1 (HIV-1) gp41. As such, it is a useful tool for probing gp41 conformations in HIV-1 membrane fusion. To establish the structural basis underlying the NC-1 specificity, X-ray crystallography was employed to solve its three-dimensional structure. To accomplish this, hybridoma-produced NC-1 antibody was first purified and digested with papain. Its Fab fragment was then purified using size-exclusion chromatography following Fc depletion using a Protein A affinity column. Finally, crystallization of NC-1 Fab was performed by the hanging-drop vapour-diffusion method and the protein was crystallized at pH 8.0 using PEG 6000 as precipitant. The results showed that the NC-1 Fab crystals belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 118.7, c = 106.0 A. There is one Fab molecule in the asymmetric unit, with 67.5% solvent content. An X-ray diffraction data set was collected at 3.2 A resolution and a clear molecular-replacement solution was obtained for solution of the structure.
Jin
Lei
L
Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA. takahashis@confsci.co.jp
Pan
Chungen
C
Qi
Zhi
Z
Zhou
Z Hong
ZH
Jiang
Shibo
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D003460:Crystallization; D018360:Crystallography, X-Ray; D015483:HIV Antibodies / Q000737:chemistry* / Q000276:immunology; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000276:immunology; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008958:Models, Molecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007140:Immunoglobulin Fab Fragments