Production, purification, crystallization and preliminary X-ray diffraction analysis of the HIV-2-neutralizing V3 loop-specific Fab fragment 7C8

Abstract

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.

Crystal Structure of the HIV-2 Neutralizing Fab Fragment 7C8 with High Specificity to the V3 Region of gp125

“…Crystals of 7C8 Fab fragments were obtained in 100 mM Tris-Cl pH 8.5, 50 mM ammonium sulphate, 25% ( w/v ) PEG 8000 and 2.5% ( v/v ) PEG 400 at 20°C by hanging-drop vapor diffusion [58]. The 7C8 crystals diffracted to a resolution of 2.7 Å and belong to space group P3 2 21 , with unit-cell parameters a  =  b  = 100.1 Å, c  = 196.8 Å.…”

“…The production, purification and crystallization of the 7C8 Fab fragments have been described previously [58] . Briefly, 7C8 was expressed from hybridomas and Fab fragments were prepared through limited digestion with papain (Sigma).…”