title: Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1.
abstract: Enteroaggregative Escherichia coli (EAEC) heat-stable enterotoxin 1 (EAST1) is a 4.1k Da protein originally discovered in EAEC but known to be scattered in other diarrheagenic E. coli as well, possibly causing diarrhea in humans and animals. We report for the first time a method to express and purify EAST1 using the Glutathione S-transferase (GST) fusion system. The gst and astA genes were fused together on a pGEX-2T plasmid vector to produce a GST-EAST1 fusion protein. Using Glutathione Sepharose affinity chromatography and C(8) reverse phase high pressure liquid chromatography, EAST1 was purified to homogeneity with a yield of 0.29 mg/L of culture. The protein purified by this method was confirmed to be EAST1 by NH(2)-terminal sequencing and mass spectrometry. The molecular weight of EAST1 is 4104.0 Da, confirming a 38 amino acid peptide as predicted by the astA gene sequence. Mass spectrometry analysis of EAST1 and of two generated peptides established the presence and suggested the position of two disulfide bridges of EAST1 in the conformations C1-C2 and C3-C4. Polyclonal antibodies were raised against EAST1 in New Zealand white rabbits to a titer of 1:8000 using affinity-purified GST-EAST1 fusion protein and to a titer of 1:100 using HPLC-purified EAST1. The biological activity of various EAST1 preparations was tested using the suckling mouse assay with CD-1 and CFW mice strains, but failed to produce fluid accumulation in the intestine.
authors.affiliation: Groupe de Recherche sur les Maladies Infectieuses du Porc (GREMIP), Faculté de Médecine Vétérinaire, Université de Montréal, C.P. 5000 Saint-Hyacinthe, Quebec J2S 7C6, Canada.
authors.forename: Louis-Philippe
authors.initials: LP
authors.lastname: Ménard
authors.forename: Jacques G
authors.initials: JG
authors.lastname: Lussier
authors.forename: François
authors.initials: F
authors.lastname: Lépine
authors.forename: Cristina
authors.initials: C
authors.lastname: Paiva de Sousa
authors.forename: J Daniel
authors.initials: JD
authors.lastname: Dubreuil
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D004768:Enterotoxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005982:Glutathione Transferase / Q000235:genetics; D013058:Mass Spectrometry; D051379:Mice; D008970:Molecular Weight; D010957:Plasmids; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D003470:Culture Media; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); D005982:Glutathione Transferase