Open AccessProduction, purification, crystallization and preliminary X‐ray diffraction analysis of the HIV‐2‐neutralizing V3 loop‐specific Fab fragment 7C8
Author(s) -
Uchtenhagen Hannes,
Sourial Samer,
Friemann Rosmarie,
Ehnlund Mariethe,
Spetz AnnaLena,
Harris Robert A.,
Madhurantakam Chaithanya,
Achour Adnane
Publication year - 2009
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309109020685
Subject(s) - crystallization , papain , monoclonal antibody , chemistry , antibody , v3 loop , microbiology and biotechnology , immunoglobulin fab fragments , ammonium sulfate , peg ratio , crystallography , biology , chromatography , biochemistry , gene , complementarity determining region , peptide sequence , enzyme , organic chemistry , finance , economics , immunology
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 m M ammonium sulfate, 100 m M 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 P 3 2 21, with unit‐cell parameters a = b = 100.1, c = 196.8 Å, and diffracted to 2.7 Å resolution.