Romano, A. (5, 14). CtxB binds to the GM1 ganglioside Mouse monoclonal to RFP Tag and can serve as a mucosal adjuvant (6, 12). Hajishengalis et al. (6) previously have shown that this A1 moiety can be replaced by a saliva-binding region (SBR) of the antigen I/II, and the chimera induced an excellent immune response to SBR when given orally to mice. A mucosal pertussis vaccine offers a number of potential advantages over the conventional parenteral vaccines, such as the ease in administration and the generation of mucosal antibodies that can prevent colonization by by ligating a 1.4-kb DNA polymerase and the primers SL155 (ATGATATCGCTTGGAGGGAAGAG; as a second gene with its authentic ribosomal binding site and leader sequence. The S1S1CtxA2B DNA was further subcloned into pMalp (New England Biolabs, Mississauga, Ontario, Canada), creating an in-frame fusion between the S1S1CtxA2 protein and the maltose binding protein. The S1S1CtxA2B fusion carried on a 1.9-kb (pMalpS1S1CtxA2B) showed that large quantities of the MBPS1S1CtxA2 fusion protein and CtxB were produced as insoluble aggregates following induction with 0.3 mM isopropyl–d-thiogalactoside. Solubilization of the aggregates with 6 M urea followed by dialysis resulted Butylated hydroxytoluene in a very low yield of the active MBPS1S1CtxA2B chimera, as indicated by GM1-binding enzyme-linked immunosorbent assay (ELISA) using anti-PT as the detecting antibody. The highest yield of the active form of the chimera was from noninduced cultures at 37C. Therefore, batches of noninduced cultures (2 liters) were used as the source (clarified sonicate) for the isolation of the chimera by affinity chromatography using an amylose column (20 ml; New England Biolabs) followed with a d-galactose column (15 ml; Pierce, Rockford, Ill.) according to the manufacturer’s suggestions. The yield of the chimera was ca. 50 g per liter of culture. The protein composition was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 12% gel and Western immunoblotting. An unheated sample of the affinity-isolated chimera showed a single band with a mass of ca. 105 kDa around Butylated hydroxytoluene the SDS-PAGE gel, suggesting purity. The boiled sample showed the disappearance of the 105-kDa band and appearance of two bands of 76 and 11 kDa. Western immunoblots showed that this 105- and 76-kDa bands were recognized by the monoclonal anti-S1 antibody (7). The 105-kDa band and eight other bands from your unheated sample were recognized by the anti-CtxB antibody (1/2,000; List Biological Laboratories, Campbell, Calif.). The multiple banding pattern is likely due to the different aggregated forms of the chimera. In the heated sample, only Butylated hydroxytoluene the 11-kDa band was recognized by the anti-CtxB antibody. The molecular mass of the native chimeric protein was estimated by gel permeation chromatography on a Sephacryl S-200HR column (2.5 by 96 cm). The eluted protein was assayed by GM1-binding ELISA. The single peak that was immunoreactive to both anti-PT and anti-CtxB antibodies has a molecular excess weight of 122,500 as estimated by comparisons to known protein standards (Sigma). The result suggests that the chimera is the expected MBPS1S1A2(CtxB)5. It is interesting that this MBPS1S1CtxA2B chimera is usually considerably larger than the holocholera toxin (84 kDa) and the SBRCtxA2B chimera (107.9 kDa ). Our results indicate that this A2 fragment can direct a polypeptide as large as 76 kDa for association with the CtxB pentamer. The functionality of the isolated chimeric protein was exhibited by GM1-binding ELISA in a dose-dependent manner. In the assay, microtiter plates were coated with 230 ng of GM1 ganglioside (CalBiochem, San Diego, Calif.) and blocked with 1% gelatin in phosphate-buffered saline (PBS) with 0.1% Tween 20 and 5 mM MgCl2, and samples were added. The chimeric protein bound to GM1 was detected with a rabbit anti-PT (1/300; ) or a goat anti-CtxB antibody followed by the goat anti-rabbit immunoglobulin G (IgG) (1/30,000; Sigma) or rabbit anti-goat IgG (1/20,000; Sigma) alkaline phosphatase conjugates, respectively. As shown in Fig. ?Fig.1,1, the titration curves in GM1-binding ELISA Butylated hydroxytoluene probed with anti-PT and anti-CtxB antibodies were very similar. The curves were also very similar to that of a commercial CtxB. Open in a separate windows FIG. 1. GM1-binding ELISA of MBPS1S1CtxA2B. The chimera was detected with a rabbit anti-PT antibody or a goat anti-CtxB antibody. Open squares represent a parallel ELISA with a commercial CtxB probed with the goat anti-CtxB antibody. The chimera did not showed any cytotoxicity to Chinese hamster ovary (CHO) cells at concentrations of below 45 g/ml in the CHO cell clustering assay (7). In comparison, the native PT was cytotoxic at 98 pg/ml. Immunogenicity of the MBPS1S1CtxA2B chimera. BALB/c mice (= 5; female;.