A Single Native Ganglioside GM(1)-Binding Site Is Sufficient for Cholera Toxin To Bind to Cells and Complete the Intoxication Pathway

Jobling, Michael G.; Yang, ZhiJie; Kam, Wendy R.; Lencer, Wayne I.; Holmes, Randall K.

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.