Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Song, Shuichuan; Xue, Jingya; Fan, Kexing; Kou, Geng; Zhou, Qian; Wang, Hao; Guo, Yajun

doi:10.1016/j.pep.2005.04.004

Cited by 21 publications

(13 citation statements)

References 18 publications

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“…Nonradioactive cell-free assays have recently been described; however, these assays require high concentrations of toxin and/or commercially purchased kits to monitor the toxin-induced inhibition of protein synthesis (18,27). A more recent cell-free method that utilizes an immuno-PCR assay for toxin detection appears to exhibit the same general level of sensitivity as our Vero-d2EGFP assay (30).…”

Section: Discussionmentioning

confidence: 99%

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Quiñones

Massey

Friedman

et al. 2009

Appl Environ Microbiol

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Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1-and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1-and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Verod2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.

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Section: Discussionmentioning

confidence: 99%

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Quiñones

Massey

Friedman

et al. 2009

Appl Environ Microbiol

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“…1-Ethyl-3-3-dimethylaminopropyl carbodiimide and N-hydroxysuccinimide were obtained from Sigma. Mut-I, PE38KDEL-I, and CD25-PE38KDEL (a control immunotoxin) were obtained as described previously (3,16). Humanized SM5-1 mAb and humanized anti-CD25 mAb were kindly provided by Shanghai Center for Cell Engineering and Antibody.…”

Section: Methodsmentioning

confidence: 99%

Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments

Gao

Kou²,

Chen³

et al. 2008

Molecular Cancer Therapeutics

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“…In this study, PE38KDEL, a 38 kDa mutant form of PE, which is a very potent toxin for preparation of immunotoxins [21,22], was used to develop PE-loaded PLGA nanoparticles. We have developed PE38KDEL-loaded PLGA nanoparticles targeting HER2 (PE-NP-HER), an important target for cancer therapy [23].…”

Section: Introductionmentioning

confidence: 99%

“…Finally, we describe the therapeutic potential of PE-NP-HER in HER2 overexpressing tumor models. Preparation of PE38KDEL and antibody fragments PE38KDEL was purified and analyzed as reported previously [21]. Briefly, expression vector pET-32a(+) containing PE38KDEL sequence was transformed into Escherichia coli strains BL21 (DE3) (Merck Biosciences) and PE38KDEL production was induced by the addition of 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) at 37°C.…”

Section: Introductionmentioning

confidence: 99%

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity

Gao

Kou

Wang

et al. 2008

Breast Cancer Res Treat

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The clinical use of Pseudomonas exotoxin A (PE)-based immunotoxins is limited by the toxicity and immunogenicity of PE. To overcome the limitations, we have developed PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with Fab' fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2). The PE38KDEL-loaded nanoparticles-anti-HER2 Fab' bioconjugates (PE-NP-HER) were constructed modularly with Fab' fragments of rhuMAbHER2 covalently linked to PLGA nanoparticles containing PE38KDEL. Compared with nontargeted nanoparticles that lack anti-HER2 Fab', PE-NP-HER specifically bound to and were sequentially internalized into HER2 overexpressing breast cancer cells, which result in significant cytotoxicity in vitro. In HER2 overexpressing tumor xenograft model system, administration of PE-NP-HER showed a superior efficacy in inhibiting tumor growth compared with PE-HER referring to PE38KDEL conjugated directly to rhuMAbHER2. Moreover, PE-NP-HER was well tolerated in mice with a higher LD(50) (LD(50) of 6.86 +/- 0.47 mg/kg vs. 2.21 +/- 0.32 mg/kg for PE-NP-HER vs. PE-HER (mean +/- SD); n = 3), and had no influence on the plasma level of plasma alanine aminotransferase (ALT) of animals when injected at a dose of 1 mg/kg where PE-HER caused significant increase of serum ALT in the treated mice. Notably, PE-NP-HER was of low immunogenicity in development of anti-PE38KDEL neutralizing antibodies and was less susceptible to inactivation by anti-PE38KDEL antibodies compared with PE-HER. This novel bioconjugate, PE-NP-HER, may represent a useful strategy for cancer treatment.

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Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Cited by 21 publications

References 18 publications

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity

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