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.
Bossé
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Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Montréal University, St-Hyacinthe, Québec, Canada.
Handl
C E
CE
Lortié
L A
LA
Harel
J
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Dubreuil
J D
JD
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics* / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D007421:Intestine, Small / Q000187:drug effects; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D001427:Bacterial Toxins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); C461599:maltose transport system, E coli