Skip to main content
Fig. 3 | Journal of Biological Engineering

Fig. 3

From: Engineered mosaic protein polymers; a simple route to multifunctional biomaterials

Fig. 3

Characterisation of Caf1 mosaic polymers by SDS-PAGE. (a) Three mutant Caf1 subunits are depicted as cartoons: Caf1WT in yellow, Caf1His in blue and Caf1A5I in orange, with a yellow star showing the location of the A5I mutation in the N-terminal β-strand that forms the subunit-subunit interface. Caf1WT: His, Caf1A5I and Caf1A5I: His proteins, as well as a mixture of Caf1A5I and Caf1WT: His, are shown in the top panel, with their expected state when heated to 70 °C shown in the bottom panel. Species which span the box represent full length Caf1 polymers. At 70 °C, the Caf1A5I subunit-subunit interactions break, leading to a pattern of oligomers (Monomer – M, Dimer – D, Trimer – T). The Caf1WT and Caf1His subunits have wild-type subunit-subunit interfaces, and do not break down at 70 °C (Polymer – P). If the Caf1A5I subunit forms mosaic heteropolymers when co-expressed with the Caf1His subunit, extra bands corresponding to oligomers containing the higher molecular weight Caf1His subunit will be present. (b) Expected gel result if co-expression of Caf1A5I: His subunits leads to mosaic heteropolymers. The expected monomer (M), dimer (D) and trimer (T) bands are shown as lines for samples 1–4 from (a) when they are heated at 70 °C. If the co-expressed subunits form separate homopolymers, samples 2, 3 and 4 would appear identical. (c) SDS-PAGE analysis of Caf1 mosaic polymers. Four samples consisting of a Caf1WT:His mosaic polymer, a Caf1A5I mutant Caf1 polymer, an Caf1A5I:His mosaic polymer and an equimolar mixture of the Caf1WT:His and Caf1A5I polymers, were heated in SDS sample buffer for 5 min at either 70 °C or 100 °C. The Caf1A5I and Caf1WT subunits have similar molecular weights (15.6 kDa), whereas the Caf1His subunit has a molecular weight which is ~ 2 kDa higher (17.2 kDa). The positions of these monomeric subunits are highlighted, and oligomeric breakdown products can be observed within the region bounded by the bracket. Oligomeric breakdown products containing the higher molecular weight Caf1His subunits in the Caf1A5I:His mosaic polymer sample are highlighted using small white triangles, with the dimer region from sample 3 expanded and shown to the left of the gel

Back to article page