N-Terminus Leader Sequence of Shiga Toxin (Stx) 1 Is Essential for Production of Active Recombinant Protein in E. coli

Oloomi, Mana; Bouzari, Saeid; Arshadi, Mania

doi:10.2174/092986606776819538

Cited by 10 publications

(8 citation statements)

References 14 publications

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“…Stx2 seems to have comparable catalytic activity to Stx1 [19]. The A and B subunits of Stx1 and Stx2 possess N-terminal signal sequences which facilitate their transport to the periplasm, where they assemble into mature toxin [20,21]. Expression of Stx is driven by a late-phase phage promoter, which is strongly activated upon induction of the bacterial SOS response.…”

Section: Introductionmentioning

confidence: 99%

Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

et al. 2013

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BackgroundShiga toxin 2 (Stx2) is a major virulence factor in gastrointestinal diseases caused by Escherichia coli. Although Stx2a (prototypical Stx2) is well-studied, all seven subtypes of Stx2 have been associated with disease in mammals. Several subtypes of Stx2, including Stx2f, are difficult to detect immunologically.Methods And FindingsFour novel monoclonal antibodies (mAbs) against the Stx2f subtype were produced and characterized. These mAbs react exclusively to the Stx2f A subunit, and do not cross-react with other subtypes of Stx2. A Stx2f-specific sandwich ELISA was established and a limit of detection of 0.123 ng/mL was obtained using one pair of the mAbs. The receptor preference of Stx2f was confirmed using this sandwich ELISA. Three out of four mAbs can partially neutralize the toxicity of Stx2f in a cell-based assay. These mAbs were also demonstrated to be highly specific and reactive when applied to colony immunoblot assays.ConclusionsNovel mAbs specific to Stx2f were developed for the first time, providing new assets for the STEC community. Immunoassays with improved sensitivity and specificity will be useful for the detection of Stx2f present in food, environmental, and clinical samples.

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Section: Introductionmentioning

confidence: 99%

Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

et al. 2013

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“…Only one pair of primers (P1 and P2) was used to amplify the whole stx1 gene and indirectly used E. coli O157:H7 as a template to amplify the entire stx1 gene. This is done rather than amplifying the genes of the subunits or initially purifying from a toxin‐converting phage, then using the toxin‐converting phage as a PCR template, which is much easier than the method of previous investigators . Furthermore, we could get final yield of the purified rStx1 ranged from 2 to 3 mg/L by one‐step nickel affinity gel column chromatography, which could be comparable yield from some previous affinity purification methods of Stx1 using globotriaosylceramide (Gb3) or egg white glycoprotein‐immobilized resins [17, 18].…”

Section: Discussionmentioning

confidence: 99%

“…showed that the presence of the N‐terminal leader sequence of the gene is essential for assembly of the subunits to yield biologically activity holotoxin (AB 5 ). The absence of the leader sequence did not affect the expression of the subunits, but it did disrupt the holotoxin assembly . However, in these studies, the construction of recombinant plasmid is more complicated.…”

Section: Introductionmentioning

confidence: 99%

A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single‐promoter vector and native signal peptide

Wang

et al. 2015

Biotech and App Biochem

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The entire stx1 region from Escherichia coli O157:H7, containing two open reading frames (stx1a and stx1b), was cloned into pET-32a with a single promoter. This region was transformed into E. coli TransB (DE3), which is a trxB and gor mutation strain. After expression in the E. coli periplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD 50 ) and median lethal dose (LD 50 ) values of approximately 30 ng and 1.5 µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3 mg/L by one-step nickel affinity gel column chromatography. This method is an easy approach to the large-scale preparation of Stx1 at a reasonable cost.

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“…Shiga toxins as holotoxins were obtained from already cloned genes in pBAD expression vector with L-arabinose; the best concentration of L-arabinose was 2% ( 13 ). The toxins were sterilized by filtering through a sterile filter (Schleicher and Schuell, Germany) and the protein concentration of Stxs was estimated by the Bradford method.…”

Section: Methodsmentioning

confidence: 99%

Comparative Effect of Recombinant Shiga Toxin in Induction of Pro- and Anti-Apoptotic Markers and Inflammatory Cytokines in Epithelial and Monocytic Cells

Jafari

Oloomi

Bouzari

2016

Jundishapur J Microbiol

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Background:Shiga toxins (Stxs, also referred to as verotoxins) are a family of bacterial protein toxins generated by Stx producing-Escherichia coli (STEC), such as E. coli serotype O157:H7.Objectives:The aim of this study was to investigate the effect of recombinant and native Shiga toxin (Stx) in induction of pro- and anti-apoptosis factors and stimulation of immune response to HeLa and THP-1 cells.Materials and Methods:The HeLa and THP-1 cells were used to study the effect of native and recombinant Shiga toxin. For this purpose, 106 cells were cultured overnight in six-well plates and different concentrations of Stx were added to each well. The cells were then collected after 24 hours of incubation. Total RNA and protein was extracted. Firstly, the total RNA was used in reverse transcription-polymerase chain reaction (RT-PCR) for detection of interleukin (IL)-1α, IL-1β, IL-8, tumor necrosis factor (TNF)-α, B-cell lymphoma (Bcl)-2 and Bcl-xl transcript. Protein expression of pro- and anti-apoptotic factors was also confirmed by western blot analysis.Results:The IL-1α and IL-8 were increased by recombinant and native Stx. Interleukin-1β was detected in THP-1, while TNF-α was detected HeLa cells. Furthermore, Bcl-2 and Bcl-xl expression was observed in HeLa cells. However, expression of Bak was reduced by recombinant Stx and native toxin at the protein level, while Bcl-xl expression was increased.Conclusions:These results suggest that toxins induce inflammatory responses, particularly through expression of chemokine. Recombinant Stx and native toxin induced apoptosis by balancing between different pro- and anti-apoptotic Bcl-2 family-factors in epithelial cells. In this study, for the first time, recombinant and native Stx induction of apoptotic factors and stimulation of immune response to HeLa and THP-1 cells were compared.

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N-Terminus Leader Sequence of Shiga Toxin (Stx) 1 Is Essential for Production of Active Recombinant Protein in E. coli

Cited by 10 publications

References 14 publications

Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

Development of Monoclonal Antibodies and Immunoassays for Sensitive and Specific Detection of Shiga Toxin Stx2f

A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single‐promoter vector and native signal peptide

Comparative Effect of Recombinant Shiga Toxin in Induction of Pro- and Anti-Apoptotic Markers and Inflammatory Cytokines in Epithelial and Monocytic Cells

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