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.
Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montreal University, St-Hyacinthe, Quebec, Canada.
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Handl
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Dubreuil
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D001427:Bacterial Toxins; D002352:Carrier Proteins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); C461599:maltose transport system, E coli