High yield of active STb enterotoxin from a fusion protein (MBP-STb) expressed in Escherichia coli.

Maltose binding protein (MBP) fused to STb, a heatstable enterotoxin of Escherichia coli, was secreted into the periplasm. A factor Xa cleavage site is present between MBP and STb allowing MBP to be cleaved from STb. The gene fusion is under the control of the strong and inducible Ptac promoter. Three hours after induction with IPTG, cells were harvested. Following osmotic shock treatment of the cells, the MBP-STb fusion protein was released and affinity-purified using an amylose resin. The fusion protein purified in this way was biologically active in ligated intestinal segments of rats. Digestion of MBP-STb with factor Xa released native STb which was purified to homogeneity by reverse-phase chromatography using a PepRPC column. The toxin was eluted at approximately 38% acetonitrile. The 5000-Da toxin was shown to be pure by SDS-PAGE and immunoblotting. The recovered enterotoxin was active in the rat loop assay. Amino acid sequence analysis showed that the first eight residues were identical to those of native STb, confirming the identity of STb. The ultraviolet absorption spectra of purified STb revealed low absorption at 254 and 280 nm compared to 210-230 nm. Isoelectric focusing under nondenaturing conditions indicated a pI of 9.6. Typically, 8 liters of bacterial culture resulted in 2.2 mg of pure STb. This genetic construction provides a readily obtainable source of biologically active STb toxin.

Fusion of the genes encoding Escherichia coli heat-stable enterotoxin b (STb) and the maltose-binding protein to obtain mature STb enterotoxin.

The heat-stable enterotoxin b gene (estB) of Escherichia coli was fused to the gene for maltose-binding protein (malE). The estB gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between malE and estB. The fused genes are controlled by Ptac, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3-4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.

Evaluation of three different STb assays and comparison of enterotoxin pattern over a five-year period in Swedish porcine Escherichia coli.

The pig intestinal loop (PIL) assay, inhibition enzyme-linked immunosorbent assay (ELISA), and DNA hybridization assay were compared for analysis of Escherichia coli heat-stable enterotoxin b (STb) on 201 porcine E. coli strains. The DNA hybridization had a 95% correlation with the STb ELISA and was therefore chosen as the method for subsequent screening of enterotoxin genes: heat labile (LT), heat-stable a (STa), and/or STb. In contrast to the PIL assay, both the STb ELISA and DNA hybridization assays were more sensitive, reliable, reproducible, and showed good correlation with each other. Consequently, the STb ELISA is preferable for analysis of toxin preparations and screening of E. coli, whereas the DNA hybridization is better for large-scale epidemiologic screening. Escherichia coli strains (n = 437) associated with porcine diarrhea isolated in Sweden during 1989 were investigated. Of the strains, 135 (31%) were positive for at least one of these toxins and, therefore, designated enterotoxigenic E. coli (ETEC). Our results were compared with the enterotoxin pattern found in earlier studies of Swedish porcine strains. The only change in occurrence of toxins was found in strains isolated from piglets less than 1 week of age. LT- and STb-producing ETEC had decreased, and STa-producing ETEC had increased in prevalence. The occurrence of STb among ETEC of weaned pigs was 93%. This toxin was also found to be more common than STa when strains from all age groups were considered.