Figure 2
From: Dodecin as carrier protein for immunizations and bioengineering applications
SEC profile of mtDod-mmACP and SDS-PAGE gel of various mtDod constructs. (a) SEC chromatogram of mtDod-mmACP after heat denaturation and DMSO precipitation. Used column: Superdex 200 increase 10/300 column (GE Healthcare). A: peak representing aggregates. D: peak representing the dodecamer. The oligomerization status was assigned based on the absorption at 375 nm (A375) and 450 nm (A450) (indicating bound flavin) and their elution volume (species at lowest molecular weight eluting with bound flavin). Although mtDod-mmACP in general formed yellow coloured aggregates, the here observed aggregate does not show flavin absorption bands, indicating that this aggregate does not contain dodecameric species. (b) SDS-PAGE gel of purified mtDod constructs. L: Ladder. For full dissociation of the dodecamers in SDS-PAGE, an acidic loading buffer containing 3.3% SDS was used during the heat treatment (5 min 60 °C). After the heat treatment, the pH was increased to about 6.8 using a glycerol- and Tris–HCl-containing buffer, followed by a second heat treatment (5 min 95 °C). MtDod-SZ1 and mtDod-SZ2 were denatured with loading buffer containing ~ 7 M urea and 2.5% SDS (prolonged heat treatment: 15 min 95 °C). Denaturation with acidic loading buffer was more reliable for most constructs and easier in handling compared to urea-based protocols (in some cases even 7–8 M urea failed to dissociate the protein completely). Of note, when treated with acidic loading buffer, some constructs showed additional bands (mainly SpyC constructs, Supplementary Fig. S5). The origin of this behaviour was not further investigated.
