FIG. 2.
Biophysical analyses of purified, CHO cell-expressed HIV-1JR-FL envelope glycoproteins. (A) Sedimentation equilibrium studies of SOS gp140 indicate that it is monomeric in solution. Representative data (open circles) obtained at a rotor speed of 9,000 rpm and a protein concentration of 0.6 mg/ml are plotted as ln(absorbance) versus the square of the axis of rotation. The slope is proportional to the molecular weight of the protein oligomer. Dashed lines with increasing slopes indicate, respectively, the predicted data for monomeric (1), dimeric (2), and trimeric (3) states of the protein. Deviations from the calculated values are plotted as residuals in the upper portion of the figure. (B) Analytical size exclusion chromatography. Purified SOS gp140, gp140UNC, and gp120 proteins were resolved on a TSK G3000SWXL column in phosphate-buffered saline, and their retention times were compared with those of known molecular mass (MW) standard proteins of 220, 440, and 880 kDa (arrowed). The main peak retention time of SOS gp140 (5.95 min) is consistent with it being a monomer that is slightly larger than monomeric gp120 (retention time, 6.24 min), whereas gp140UNC (retention time, 4.91 min) migrates as an oligomeric species. (C) The oligomeric status of pure standard proteins, thyroglobulin, ferritin, and albumin, were compared with gp120 and gp120 in complex with soluble CD4 using BN-PAGE. The proteins were visualized on the gel using Coomassie blue. (D) BN-PAGE analysis of CHO cell-derived, purified HIV-1JR-FL gp120, SOS gp140, and gp140UNC glycoproteins.