Fig. 3

Localisation of FLAG-tagged and sortase signal-linked CipC3 miniscaffoldin variants when produced alone and when co-produced with S. aureus SrtA (Sa-SrtA). 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: wild-type C. acetobutylicum ATCC 824 (WT); CEL01, expressing a gene encoding CipC3-FLAG; CEL03, expressing a gene encoding the internally FLAG-tagged variant CipC2F3; CEL04, expressing a gene encoding CipC2F3-CA_C0353ss; CEL05, expressing a gene encoding CipC2F3-CA_C0205ss; and CEL06, expressing genes encoding CipC2F3-CA_C0205ss and Sa-SrtA. Sa-SrtA was expected to recognise the sortase signal sequence of CA_C0205, but was not expected to be able to anchor the protein to the cell wall, and correspondingly, cell wall anchoring of CipC2F3-CA_0205ss is absent in CEL06. L, ColorPlus Prestained Protein Ladder, broad range (10–230 kDa); +, carboxy terminal FLAG-BAP control protein (50 kDa)