Expression, purification, and biochemical characterization of enteroaggregative Escherichia coli heat-stable enterotoxin 1

Ménard, Louis-Philippe; Lussier, Jacques G.; Lépine, François; Sousa, Cristina Paiva de; Dubreuil, J. Daniel

doi:10.1016/j.pep.2003.09.008

Cited by 31 publications

(27 citation statements)

References 22 publications

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“…On the contrary to what was determined for human EAST1-positive strains, there is no clear evidence of EAST1-dependent fluid secretion in animal studies [22] or of an association of EAST1 with epidemic strains of E. coli with high diarrheagenic potential. The late purification of EAST1 toxin [24] by our group could provide a source of pure toxin in order to test if diarrhea could result from the administration of pure toxin to animals without the cumbersome interpretation that we encounter when using E. coli isolates or crude culture supernatants. Another challenge for future studies will be to investigate if the numerous variants already reported for EAST1 toxin are actually toxic for farm animals and also to determine their association with diarrhea outbreaks occurring in farm animals.…”

Section: Discussionmentioning

confidence: 99%

“…Unlike STa, a classic signal peptide was not observed in the N-terminus of the predicted EAST1 sequence. Most information on this toxin was acquired through the use of culture filtrates or by direct use of EAST1-producing strains since EAST1 was purified only recently [24]. Culture filtrates from EAEC strain 17-2, heated at 65 °C for 15 min, has demonstrated partial heat stability.…”

Section: East1 Toxinmentioning

confidence: 99%

“…Culture filtrates from EAEC strain 17-2, heated at 65 °C for 15 min, has demonstrated partial heat stability. Among the 38 amino acids composing EAST1, there are four cysteines at positions 17, 20, 24, and 27, which have been shown recently to form two disulfide bridges [24]. Indirect evidence of the importance for biological activity of the disulfide bridges formed between cysteines 17 and 27 was obtained when cysteine 17 was replaced by an alanine: it resulted in inactivation of EAST1 [23].…”

Section: East1 Toxinmentioning

confidence: 99%

“…EAST1 is a peptide of 38 amino acids with a molecular weight of 4.1 kDa. The four cysteines of the toxin are involved in the formation of two disulfide bridges [24]. Further studies associated EAST1 with E. coli pathotypes other than EAEC, such as EHEC, ETEC, DAEC and EPEC, and also with other bacterial genera like Salmonella [36].…”

Section: Introductionmentioning

confidence: 95%

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Presence of Escherichia coli carrying the EAST1 toxin gene in farm animals

Veilleux

Dubreuil

2006

Vet. Res.

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-Enteroaggregative Escherichia coli (EAEC) heat-stable toxin 1 (EAST1) is a small toxin of proteic nature. The reference strain producing this toxin was originally detected in the stools of a diarrheic Chilean child. Today, we know that EAST1 is not solely associated with EAEC but also with many other diarrheic E. coli families. Some studies have established the role of EAST1 in human outbreaks of diarrhea. In addition, isolates from farm animals were shown, more recently, to carry the astA gene coding for EAST1. However, the relation between the presence of EAST1 and disease is not conclusive. In this article, the current state of the knowledge on the presence and probable role of EAST1 in farm animal diseases is reviewed and discussed.

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Section: Discussionmentioning

confidence: 99%

Section: East1 Toxinmentioning

confidence: 99%

Section: East1 Toxinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

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Presence of Escherichia coli carrying the EAST1 toxin gene in farm animals

Veilleux

Dubreuil

2006

Vet. Res.

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“…Therefore, the expression and the preparation of recombinant mPNAS-4 protein (rmPNAS-4) and its polyclonal antibody should provide effective experimental tools for identifying its subcellular localization, expression profile, the interacting proteins, etc. This strategy of studying novel genes at the protein level should facilitate further investigations and is generally accepted worldwide (3)(4)(5)(6)(7)(8).…”

Section: Introductionmentioning

confidence: 99%

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Zhang

Wang

Yan

et al. 2008

Braz J Med Biol Res

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Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

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