FIG 11.
EM and SPR analyses of variants of the 92UG037.8 gp140UNC-Fd-His proteins containing flexible linkers. The 92UG037.8 gp140-FL20-His, gp140-FL20-IP-His, and gp140-FL20-SOSIP.664-His constructs are shown in Fig. 1. (A to C) The proteins were purified by the Ni-NTA/SEC method or via a PGT145 bNAb affinity column as indicated and then analyzed by EM (A) or SPR (B and C). (A) The gp140-FL20-His proteins were purified by Ni-NTA/SEC. Attempts to purify native-like trimers based on this construct via the PGT145 column did not yield sufficient protein for EM analysis. The gp140-FL20-IP-His (middle row) and gp140-FL20-SOSIP-His (bottom row) proteins were purified by Ni-NTA/SEC or by the PGT145 affinity column; compared to the Ni-NTA/SEC method, the recoveries of trimers from the PGT145 column were ∼5% and ∼25%, respectively. (B) SPR data are shown for all three variants of the 92UG037.8 gp140-FL20-His construct, each purified by Ni-NTA/SEC. Each variant is depicted in a different color, as indicated. The quaternary epitope-specific bNAbs PG16 and PGT145 (trimer apex) and 35O22 (gp120-gp41ECTO interface) serve as diagnostic antibodies for assessing the native-like trimer content. The CD4bs non-NAb F105 was used to assess the nonnative Env content of the samples. (C) SPR data were derived using gp140-FL20-SOSIP-His proteins purified either by Ni-NTA/SEC (green) or by a PGT145 affinity column (gray). The PGT145-purified SOSIP.664-His trimer (blue) serves as a comparator for each test antibody.