Fig. 6

Design and production of a 3-subunit mosaic Caf1 polymer. (a) Diagram depicting the pBad-Caf1^OPN and pBad2x-Caf1^OPN:BMP2 plasmids used in this study. The pBad plasmid was used as a template for the insertion of a caf1 mutant gene containing the osteopontin sequence (Caf1^OPN, yellow), a second ribosome binding site (RBS, green) and a caf1 mutant gene containing the BMP2 peptide sequence (Caf1^BMP2, blue) in order to construct the pBad2x-Caf1^OPN:BMP2 plasmid, which was thus designed to express two caf1 genes at once under the control of the arabinose inducible promoter. The pBad-Caf1^OPN plasmid, designed to express only the one caf1 gene, is shown alongside as a comparison. (b) SDS-PAGE analysis showing the expression of a 3-subunit mosaic Caf1 polymer. Cultures of E. coli BL21(DE3) cells transformed with pCOP and either pBad-Caf1^OPN, pBad-Caf1^BMP2 or pBad2x ^OPN:BMP2 were grown for 22 h in the presence and absence of 1% w/v arabinose. Samples of the extracellular fraction (flocculent layer and supernatant) were then heated to 100 °C for 5 min in SDS containing buffer and applied to the gel. The “U” lane represents the pCOP/pBad2x-Caf1^OPN:BMP2 1% arabinose sample that was not heated to 100 °C before application, and shows most Caf1 subunits are present in high molecular weight polymers. The monomeric subunits corresponding to each mutant are shown by numbers next to the relevant band: 1 is Caf1^WT, 2 is Caf1^OPN and 3 is Caf1^BMP2