Expression of CipA scaffoldin variants driven by an additional promoter. Supernatants of CipA-expressing recombinant C. acetobutylicum strains (CipA2, CipA3, thlCipA2, thlCipA3, CipA_BB2, CipA_DNA2.0, thl_CipA_BB2, thl_CipA_DNA2.0) were TCA precipitated and concentrated 100-fold. ANTI-FLAG M2 monoclonal antibody-horseradish peroxidise conjugate (Sigma) was used to detect the Flag-tagged CipA variants. M, ColorPlus Prestained Protein Ladder (10–230 kDa; New England Biolabs). The N-Flag-Bap control protein (50 kDa, 50 μg/lane; Sigma) was used to estimate the amount of secreted CipA variants. Expected molecular masses are indicated by arrows.