Mechanisms of Assembly and Cellular Interactions for the Bacterial Genotoxin CDT
Figure 6
Cation-Exchange Chromatography of CDT Holotoxin Containing N- and C-Terminal Deletions of CdtC
Wild-type (A) or mutant CDT holotoxins [(B) holotoxin with CdtC (Δ21−35), (C) holotoxin with CdtC (Δ21−39), (D) holotoxin with CdtC (Δ179−186)] were run on Fast Flow SP Sepharose columns (20 ml) using an ÄKTA FPLC. Refolded CDT complexes were loaded on an SP Sepharose column in buffer containing 40 mM NaCl, 20 mM HEPES (pH 7.5), 2.5 mM DTT and analyzed by a gradient in salt concentration as described in Materials and Methods. Individual fractions (5 ml) or pooled material (P) from elution peak were collected and examined by SDS-PAGE. Proteins were stained with Coomassie blue dye. Images of the gels are presented inside corresponding chromatograms. The peak that elutes between 100 and 150 mM NaCl concentration (P1) contains an intact CDT holotoxin, and the second peak (P2) that elutes at concentrations higher than 200 mM NaCl contains only the CdtB subunit. A, CdtA; B, CdtB; C, CdtC; C*, deletion mutants of CdtC; L, loaded material; fr, fraction number; Fl, flowthrough; P, pooled material from elution peak. Absorbance was measured at 280 nm. In A, P1 contains fractions 14−22, and P2 contains fractions 31−34; in D, P1 contains fractions 20−22, and P2 contains fractions 32−34.