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. 2013 Oct 3;9(10):e1003644. doi: 10.1371/journal.ppat.1003644

Figure 6. The N-terminus of the B-subunit is required for normal B+C sub-complex synthesis and secretion.

Figure 6

(A) Arabinose inducible E. coli heterologous production constructions encoding full length or N-terminal truncated tcdB1 with tccC5 containing C-terminal FLAG-tag fusions. Construct “BC5” encodes full-length tcdB1 and tagged tccC5, “BΔC” encodes an N-terminal truncated copy of tcdB1 and a tagged tccC5, “B” encodes full-length tcdB1 only and “C5” encodes the tagged tccC5 only. “V” represents pBAD30 vector only control. All constructs use the native translation initiation regions. (B) Western blot analysis of concentrated supernatants, membrane (inner and outer) and soluble fractions (cytoplasm and periplasmic contents) of these induced constructs using an anti-peptide antibody that was raised against the C-terminus of B-subunit. (C) Western blot analysis of these same samples using an anti-FLAG tag antibody. The asterisk and arrow indicators are discussed in the text. An anti β-lactamase (Anti-Bla) Western blot is included to provide a control for fractionation sample quality and loading amount.