Figure 3

Biochemical characterization of cell line-derived BG505 SOSIP.664 gp140 trimers and comparator proteins. (A) BN-PAGE analysis of 2G12-purified Env proteins produced by the BG505 SOSIP.664-expressing 293 T and CHO stable cell lines. The gels were stained with Coomassie blue. The molecular weights of marker (M) proteins (thyroglobulin, 669 kDa and ferritin, 440 kDa) are indicated. (B) A similar BN-PAGE analysis, but of SEC-purified trimers. (C) SDS-PAGE analysis of SEC-purified BG505 SOSIP.664 trimers, under non-reducing (- DTT) and reducing (+ DTT) conditions, followed by Coomassie blue staining. Cleavage of gp120 from gp41_ECTO is assessed by the conversion of the gp140 band to gp120 in the presence of DTT; the released gp41_ECTO subunit is not always stained strongly. (D) Western blot analysis of a reducing SDS-PAGE gel to assess the quality of SEC-purified BG505 SOSIP.664 trimers. The blots were probed with the anti-gp120 MAb, ARP 3119. No Env degradation products arising from V3-clipping or other proteolysis events are visible. (E) Reducing SDS-PAGE analysis of SEC-purified BG505 SOSIP.664 trimers and comparator Env proteins, followed by Coomassie blue staining. The comparator proteins were also SEC-purified to yield the 120-kDa fraction for MN gp120 and the “trimer” fraction (i.e., proteins containing three gp120 and three gp41_ECTO subunits) for uncleaved CZA97.012 gp140. Multiple degradation and/or aggregation products derived from MN gp120 and CZA97.012 gp140 are visible, but none from the BG505 SOSIP.664 trimers. (F) Western blot analysis of the same gel shown in (E). The detection antibodies were a pool (1:1:1) of polyclonal HIV-Igs derived from subtype A, B and C infections. The various aggregates or degradation products seen in (E) are Env-based.