Production and purification of cholera toxin variants with 0, 1, 2, 3, 4, or 5 native GM 1 binding sites. To produce CT holotoxins with defined combinations of wt and mutant CTB subunits, we made three compatible plasmid constructs encoding either tagged wt CTB, CTB-G33D, or wt CTA and expressed them in the same Escherichia coli strain to produce a holotoxin pool with B pentamers consisting of tagged wt CTB and/or CTB-G33D, from which to purify all six possible holotoxin variants. The tag is a 34-aminoacid peptide (denoted GSH6) which is genetically appended after the Met103 codon of native ctxB and encodes glycosylation (bold) and sulfation sites (underlined) (24) , SSSGGGGSSH-PNNTSNNTSSAEDYEYPS, followed by six His residues. Each plasmid has a different replication origin, antibiotic selection, and combination of promoters ( Fig. 1A ; Table 1 ). The ctxA gene, encoding CTA, is expressed from dual pLac and pBAD promoters. Holotoxins and free B pentamers were purified from whole-cell lysates by metal-ion affinity chromatography on Talon resin, and free B pentamers were removed by passing the eluate pool over cation exchange resin, resulting in binding of the free CTB pentamers to the resin and recovery of holotoxin in the flowthrough (Fig. 1B) . Holotoxins with different numbers of BS were then separated by anion-exchange chromatography (Fig. 1C) . By varying the amount of each inducer, we sought to alter the ratio of wt and mutant B subunits in the holotoxin pool. However, for practical purposes, we found that a single condition of relatively low levels of arabinose (0.0005% to express GSH6-tagged wt-CTB) and high levels of isopropyl-␤-d-thiogalactopyranoside (IPTG) (400 M to express native-size CTB-G33D) gave acceptable yields of holotoxin with homopentameric and singly or doubly tagged heteropentameric B subunits (with zero, one, and two wt BS, respectively) and detectable but lower yields of the triple, quadruple, and homopentameric tagged B subunits as described in detail in the next paragraph. This production strain we designated AMBT (for A, mutant CTB, wt CTB-tagged). Simply by swapping the wt and G33D alleles in the respective CTB-encoding vectors and keeping the same inducer ratios, we were able to express the single or double tagged heteropentameric and homopentameric wt CTB subunits with three, four, and five wt BS, respectively, from the production strain designated ABMT (A, wt CTB, mutant CTBtagged). Using these strategies, from two production strains, we produced all six variant holotoxins, which had from zero to five wt BS and a maximum of two tagged subunits. A third production strain, designated ABBT (A, wt CTB, wt CTB-tagged), in which both the GSH6-tagged and untagged B polypeptides were wt CTB, was used under the same expression conditions to produce control holotoxins with five native BS and zero, one, or two tagged CTB subunits. Figure 1 shows the purification process for the AMBT strain (mutant CTB-G33D, wt CTB tagged). Since CTB pentamers naturally bind to metal affinity resins (25) , cholera toxin variants can be purified to near-homogeneity by a single passage over Talon resin. The imidazole eluate pool from the Talon resin contained a mixture of holotoxin and some free CTB pentamers. These free pentamers were removed by cation exchange chromatography, where at pH 8.0 in 50 mM Tris buffer, holotoxin passed through the column while free pentamers bound and could be eluted subsequently with a salt gradient (Fig. 1B) . The heterogenous mixture of holotoxins was further separated into its components by anion-exchange chromatography. The GSH6 tag not only changed the size of the B monomer but also changed its predicted pI from 8.24 for native CTB to 6.21 for CTB-GSH6. The respective predicted pIs for the CTB-G33D variant changed from 7.31 for the native length to 5.91 for the GSH6-tagged variant. All holotoxins bound to the anion exchange matrix, and at least five peaks could be discerned following elution with a salt gradient (Fig. 1C) . Samples from individual fractions were analyzed by SDS-PAGE either without (Fig. 1D , upper) or after (Fig. 1D , lower) boiling in sample loading buffer. The unboiled samples in lanes 4 to 13 showed a single band corresponding to assembled holotoxin (AB 5 ), with a small amount of contaminating protein of slower mobility in lanes 2 and 3. The major fractions from each peak are homogenous and are consistent with each peak containing a single species CTB-G33D-Linker-His 6 , pAra BAD , Cm r , Ori p15a This study of variant holotoxin, The assembled holotoxins in the unboiled samples bind less SDS than if they were fully denatured and therefore do not migrate in direct proportion to their molecular weights. The relative molecular weight (M r ) for each holotoxin variant is increased by the presence of one or more tagged CTB subunits. Each of these holotoxins completely disassembles into its component polypeptides after boiling (Fig. 1D , lower panel; CTA, 29 kDa; CTB-GSH6-tagged monomer, 14.4 kDa; and native CTB-G33D monomer, 11.5 kDa). The holotoxin in peak 0 ( Fig. 1C ) has homopentameric CTB-G33D subunits and no functional GM 1 BS, while holotoxins in peaks 1 and 2 have a mixture of wt CTB-GSH6 and CTB-G33D pentamers with 1 and 2 GM 1 BS, respectively. The fractions loaded in lanes 2 to 5 contain CTA plus 5 CTB-G33D (peak 0), and those in lanes 6 to 9 contain CTA plus 1 wt CTB-GSH6 and 4 CTB-G33D (peak 1), and those in lanes 10 to 13 contain CTA plus 2 wt CTB-GSH6 and 3 CTB-G33D (peak 2). Only these first three peaks were well enough separated to be purified efficiently from the extract of production strain AMBT. The fractions corresponding to each peak were pooled, dialyzed, and rerun over the same anion-exchange column to achieve a very high degree of purity. To make holotoxins with 3 and 4 native GM 1 BS, an expression and purification run was done using strain ABMT, which produced three peaks similar to those shown in Fig. 1C , containing holotoxins with zero, one, or two GSH6tagged CTB-G33D subunits and five, four, or three native GM 1 BS, respectively. Finally, as noted above, strain ABBT was used for expression and purification of wt control holotoxins, all with 5 GM 1 BS and zero, one, or two GSH6-tagged CTB subunits. Analysis of the eight final highly purified holotoxin preparations by SDS-PAGE and Coomassie blue staining is shown in Fig. 2 . The upper panel shows a gel run with unboiled samples, and the lower panel shows a gel run with samples boiled in sample buffer to dissociate the components. Without boiling, holotoxins with untagged or tagged wt CTB pentamers (Fig. 2 , upper panel, lanes 2 to 4) dissociated into free CTA and stable pentameric B subunits that ran near the 45-kDa marker (lane 1). Interestingly, the presence of one or more CTB-G33D polypeptides increased stability of the heterohexameric AB 5 holotoxins. Consequently, unboiled holotoxins with three or more CTB-G33D polypeptides (Fig. 2 , upper panel, lanes 5 to 7) remained fully associated and ran near the 66-kDa marker (lane 1), whereas unboiled holotoxins with one or two CTB-G33D subunits exhibited partial dissociation into B pentamer and free CTA (Fig. 2 , upper panel, lanes 8 and 9). The high stability of the heterohexameric AB 5 holotoxins containing five, four, or three CTB-G33D polypeptides is also evident in the upper panel of Fig. 1D . As expected, increasing the number of tagged CTB polypeptides resulted in moderate decreases in observed mobilities of the pentamer bands (Fig. 2 , upper panel, compare lanes 2, 3, and 4) and the holotoxin bands (Fig. 2 , upper panel, compare lanes 5, 6, and 7), and increasing the number of CTB-G33D polypeptides resulted in slight increases in mobilities of the holotoxin bands (Fig. 2 , upper panel, compare lanes 6 to 8 and lanes 7 to 9). Upon boiling, all holotoxins dissociated and resolved into their individual polypeptide components (Fig. 2, lower panel) . All holotoxins have one CTA subunit. Holotoxins in lanes 2 and 5 are predicted to have five native CTB monomers; holotoxins in lanes 3, 6, and 8 have are predicted to have one tagged CTB and 4 untagged CTB monomers; and holotoxins in lanes 4, 7, and 9 are predicted to have two tagged and three untagged CTB monomers.