Figure 8From: Secretion and assembly of functional mini-cellulosomes from synthetic chromosomal operons in Clostridium acetobutylicum ATCC 824Native PAGE and western blot analysis of cellulosomal operons assembled in vivo by the recombinant C. acetobutylicum strains. The culture supernatants of the recombinant strains (thlOIDCel9B, thlOIDCel8A, thlOIDCel9B_CipA3, thlOIDCel8A_CipA3, thlOIDCel9B_Cel8A_CipA3, thlOIDCipA3) were concentrated 70-fold by ultracentrifugation and 10 μl of the concentrated samples loaded onto a native PAGE Tris-acetate gel. The presence of the cellulosomal subunits and the in vivo assembly of the mini-cellulosomes was detected using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma). The presence of multiple bands in the samples expressing mini-cellulosomes (thlOIDCel9B_CipA3, thlOIDCel8A_CipA3 and thlOIDCel9B_Cel8A_CipA3) likely represents unsaturated complexes with cellulases binding different cohesin domains. These samples were loaded at two different concentrations: 10 and 5 μl (½) of the original samples.Back to article page