Fig. 4

Coexpression of genes encoding sortases with a sortase signal-linked scaffoldin. Cells were fractionated according to the protocol in the “Methods” section, and fractions were subjected to SDS-PAGE and western blot. Proteins were separated on 4–12 % Bis–Tris gradient gels; after blotting, cellulosomal components were detected using ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate. Four fractions were examined: SN culture supernatant; CW cell wall digest; PP protoplast fraction; WC whole cell fraction. Six different strains were examined: C. acetobutylicum ATCC 824 wild-type (WT); CEL03, expressing a gene encoding CipC2F3; CEL05, expressing a gene encoding CipC2F3-CA_C0205ss, and CEL08, CEL09, and CEL10, combining production of CipC2F3-CA_C0205ss with overexpression of the srtA genes of C. acetobutylicum, L. monocytogenes, and B. cereus, respectively. L, ColorPlus Prestained Protein Ladder, broad range (10–230 kDa); +, carboxy terminal FLAG-BAP control protein (50 kDa)