Open AccessFab crystallization and preliminary X‐ray analysis of NC‐1, an anti‐HIV‐1 antibody that recognizes the six‐helix bundle core of gp41
Author(s) -
Jin Lei,
Pan Chungen,
Qi Zhi,
Zhou Z. Hong,
Jiang Shibo
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309110019287
Subject(s) - immunoglobulin fab fragments , gp41 , crystallization , crystallography , chemistry , monoclonal antibody , molecular replacement , molecule , helix bundle , helix (gastropod) , antibody , crystal structure , protein structure , biology , biochemistry , epitope , organic chemistry , ecology , snail , immunology , complementarity determining region
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 P 3 2 21, with unit‐cell parameters a = b = 118.7, c = 106.0 Å. 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 Å resolution and a clear molecular‐replacement solution was obtained for solution of the structure.