Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit.
The 3' terminus of the DNA coding for the extracellular Escherichia coli heat-stable enterotoxin (ST) devoid of transcription and translation stop signals was fused to the 5' terminus of the DNA coding for the periplasmic B subunit of the heat-labile enterotoxin (LTB) deleted of ribosomal binding sites and leader peptide. By RNA-DNA hybridization analysis, it was shown that the fused DNA was transcribed in vivo into an RNA species in close agreement with the expected molecular weight inferred from the nucleotide sequence. The translation products of the fused DNA resulted in a hybrid molecule recognized in Western blots (immunoblots) with antibodies directed against the heat-labile moiety. Anti-LTB antibodies coupled to a solid support bound ST and LTB simultaneously when incubated with ST-LTB cellular extracts. By [35S]cysteine pulse-chase experiments, it was shown that the fused ST-LTB polypeptide was converted from a precursor with an equivalent electrophoretic mobility of 20,800 daltons to an approximately 18,500-dalton species, which accumulated within the cell. The data suggest that wild-type ST undergoes at least two processing steps during its export to the culture supernatant. Blocking the natural carboxy terminus of ST inhibited the second proteolytic step and extracellular delivery of the hybrid molecule.
Guzman-Verduzco
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LM
Department of Microbiology, University of Texas Health Science Center, Dallas 75235.
Kupersztoch
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YM
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D008970:Molecular Weight; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); C027538:heat-labile enterotoxin, E coli