Fig. 6: p53 folding and tetramerization using tES–F116H.
From: A general approach to protein folding using thermostable exoshells
a AUC demonstrates tES–F116H(+/−) and tES–F116H(+) encapsulated a single p53 monomer. b p53 release from tES–F116H(+/−) and tES–F116H(+) results in tetramer formation as demonstrated by AUC. The use of tES–F116H(–) results in aggregation. c SDS gel demonstrates monomeric p53 in the tES:POI complex, and tetrameric p53 after monomer release. Lane 2 shows the tES subunits. Prior to tES release, p53 monomer and tES–F116H(+/−) subunits were mixed (pH 8.0) and visualized using SDS-PAGE as a 47 kDa and 20 kDa (lane 6). After tES release, p53 was predominantly seen as an ~180-kDa band that was attributed to the tetramer (lane 4). Upon denaturation (boiling with reducing reagents) of the same sample, the tetramer band disappeared and a 47-kDa monomer band was visible (lane 3). When p53 monomer was folded without tES, it failed to tetramerize (lane 7). In lane 5, 50 μM EDTA is added and p53 tetramers partially break into monomers and dimers (lane 5) (n = 3 independent experiments). [Note: CB—cage break] (d), tES–F116H(+/−) (e), tES–F116H(+) (f), tES–F116H(–), (g), p53 folded without tES. p53 folded with tES–F116H(+/−) shows a characteristic tetramer denaturation peak at 50 °C. Addition of mAb1620 results in a novel complex peak at ~95 °C. Differential scanning fluorimetry of mAb240, and p53 and h, tES–F116H(+/−) i, tES-F116H(+) j, tES–F116H(–) k, p53 folded without tES. p53 denaturation is affected by conA (l) but not a scrambled sequence (m). AUC analysis of tES–F116H(+/−)-folded tetramers demonstrates complex formation in the presence of ConA (n) but not in the presence of scr-conA (o).
