FIGURE 1.

Expression and purification of intact ACT and domains. A, adenylate cyclase toxin domain architecture. ACT is a 177-kDa protein toxin, consisting of five sequential domains as follows: the catalytically active N-terminal adenylate cyclase (CAT) domain, the central hydrophobic (HP) domain, modification (Mod) region carrying two acylation sites, Lys-860 and Lys-983, the RTX domain, and finally a C-terminal secretion signal (Sec). The five shaded blocks in the RTX region represent tandem Gly-Asp-rich repeats. B, ACT forms high molecular mass species (>600 kDa) when urea is removed by dialysis or dilution. Size exclusion chromatograms of ∼120 μg of ACT using a Superdex 200 column were collected directly after a 1:10 dilution (final urea concentration 0.8 m) or overnight dialysis into HBSC with 1 m urea. Arrow indicates the size expected for monomer, 177 kDa. C, SDS-polyacrylamide gel comparing full-length ACT with different domain constructs after purification from E. coli. Three versions of the CAT domain (residues 1–373 (CAT₃₇₃), 1–385 (CAT₃₈₅), and 1–400 (CAT₄₀₀)) and three versions of the RTX domain (residues 985–1706 (RTX₉₈₅), 751–1706 (RTX₇₅₁), and acylated 751–1706 (RTX₇₅₁*)) were expressed from plasmid pET28a with an N-terminal His₆ tag. The HP domains (residues 399–1096 (HP₁₀₉₆) and acylated 399–1096 (HP₁₀₉₆*)) were expressed from plasmid pMalc-5x with an N-terminal MBP fusion for enhanced solubility and C-terminal His₆ tag.