Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8.
7C8 is a mouse monoclonal antibody that is specific for the third hypervariable loop (V3 loop) of the human immunodeficiency virus type 2 (HIV-2) associated protein gp125. Fab fragments of 7C8 effectively neutralize HIV-2. 7C8 was expressed and purified from a hybridoma cell line in order to establish the molecular basis underlying the specificity of the 7C8 antibody for the V3 loop as well as the specific role of the elongated third complementarity-determining region of the heavy chain (CDRH3). The antibody was digested with papain and Fab fragments were purified using size-exclusion chromatography. Hanging-drop vapour-diffusion crystallization techniques were employed and the protein was crystallized in 50 mM ammonium sulfate, 100 mM Tris-HCl pH 8.5, 25%(w/v) PEG 8000 and 2.5%(w/v) PEG 400 at 275 K. The analysed crystals belonged to the rhombohedral space group P3(2)21, with unit-cell parameters a = b = 100.1, c = 196.8 A, and diffracted to 2.7 A resolution.
Uchtenhagen
Hannes
H
Center for Infectious Medicine (CIM), F59 Department of Medicine, Karolinska University Hospital Huddinge, Karolinska Institutet, SE-141 86 Stockholm, Sweden.
Sourial
Samer
S
Friemann
Rosmarie
R
Ehnlund
Mariethe
M
Spetz
Anna-Lena
AL
Harris
Robert A
RA
Madhurantakam
Chaithanya
C
Achour
Adnane
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification*; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D015498:HIV-2 / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000302:isolation & purification*; D051379:Mice; D009500:Neutralization Tests; D017433:Protein Structure, Secondary
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments