For several years, tools to study the conformational changes of HIV-1 envelope glycoproteins have been developed in order to comprehend those changes and their role in the fusion process and immunogenicity of HIV-1. To facilitate these studies, expression of the HIV-1 gp120 envelope glycoprotein has been done in several over-expression settings. However, over-expression of HIV-1 gp120 in mammalian cells leads to the formation of aberrant disulfide-linked dimers that can bias the results of experiments aimed at measuring gp120 affinity with different ligands. The presence of these gp120 dimers, generated in a widely used gp120 expression system, affects the affinity of gp120 for CD4-induced ligands, as evaluated by surface plasmon resonance. Upon monomeric gp120 purification, neither the removal of potential glycosylation sites on V4 nor the removal of the V5 variable region affect the overall affinity of gp120 for 17b and A32 CD4-induced ligands. Removal of these aberrant disulfide-linked gp120 dimers by standard size exclusion chromatography is sufficient to restore the overall affinity of gp120 preparations for these ligands.
Keywords: CD4-induced epitopes; FPLC; HIV; Surface plasmon resonance; gp120; gp120 dimers.
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