Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa.

Gray, Gregory L.; Smith, Douglas H.; Baldridge, Jane S.; Harkins, Richard N.; Vasil, Michael L.; Chen, Ellson Y.; Heyneker, Herbert L.

doi:10.1073/pnas.81.9.2645

Cited by 250 publications

(125 citation statements)

References 31 publications

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“…Cloning, Expression, and Purification of Exotoxin A-The coding sequence for PE (GenBank TM accession number AAB59097) was amplified by PCR from P. aeruginosa genomic DNA (18) and cloned into pMMB67H vector using HindIII and EcoRI restriction sites. Alternatively, PE that carries a sortase recognition motif, LPETG, near its C terminus followed by His 6 was cloned into pMMB67H vector using the same restriction enzymes.…”

Section: Methodsmentioning

confidence: 99%

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

Tafesse

Guimarães

Maruyama

et al. 2014

Journal of Biological Chemistry

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Background: Bacterial toxins, including P. aeruginosa exotoxin A (PE), are valuable tools to dissect biological processes. Results: A genome-wide genetic screen identifies several novel host factors used by PE, including GPR107. Conclusion: Bacterial toxins can help identify novel host components involved in key intracellular trafficking steps. Significance: GPR107 may be a receptor that associates with G-proteins at the Golgi to regulate membrane transport.

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Section: Methodsmentioning

confidence: 99%

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

Tafesse

Guimarães

Maruyama

et al. 2014

Journal of Biological Chemistry

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“…When the wild type gene for ToxA has been expressed by Escherichia coli , various results have been achieved ranging from no secretion (Gray et al, 1984), periplasmic secretion (Lory et al, 1988) to extracellular secretion using an OmpA signal sequence substituted for that of the ToxA signal sequence (Chaudhary et al, 1988). Among the different chimeric proteins, TGFα-PE40 can be recovered from inclusion bodies, the periplasm, and supernatant when used with the OmpA signal sequence (Kondo et al, 1988), while PE35/TGFαKDEL was only found in inclusion bodies.…”

Section: Introductionmentioning

confidence: 99%

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Quintero

Carrafa²,

Vincent³

et al. 2016

Biotech & Bioengineering

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Tumor-targeted Salmonella VNP20009 preferentially replicate within tumor tissue and partially suppress tumor growth in murine tumor models. These Salmonella have the ability to locally induce apoptosis when they are in direct contact with cancer cells but they lack significant bystander killing, which may correlate with their overall lack of antitumor activity in human clinical studies. In order to compensate for this deficiency without enhancing overall toxicity, we engineered the bacteria to express epidermal growth factor receptor (EGFR)-targeted cytotoxic proteins that are released into the extracellular milieu. In this study, we demonstrate the ability of the Salmonella strain VNP20009 to produce three different forms of the Pseudomonas exotoxin A (ToxA) chimeric with a tumor growth factor alpha (TGFα) which results in its producing culture supernatants that are cytotoxic and induce apoptosis in EGFR positive cancer cells as measured by the tetrazolium dye reduction, and Rhodamine 123 and JC-10 mitochondrial depolarization assays. In addition, exchange of the ToxA REDLK endoplasmic reticulum retention signal for KDEL and co-expression of the ColE3 lysis protein resulted in an overall increased cytotoxicity compared to the wild type toxin. This approach has the potential to significantly enhance the antitumor activity of VNP20009 while maintaining its previously established safety profile.

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“…The gene encoding exotoxin A from P. aeruginosa PA103, a hyper-toxin-producing isolate (22), has been cloned and its nucleotide sequence has been determined (11). In addition, the crystalline structure of exotoxin A revealed three structural domains (1).…”

mentioning

confidence: 99%

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1

Wick

Iglewski

1988

J Bacteriol

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Analysis of purified exotoxin A from parental Pseudomonas aeruginosa PAO1 and mutant strain PAO-PR1, which produces enzymatically inactive exotoxin A (CRM 66), revealed that CRM 66 lost 90% of parental enzymatic activity. Nucleotide sequence analysis of cloned exotoxin A genes showed a single amino acid substitution in CRM 66. Position 426 in the mature protein of parental (PAO1) exotoxin A is histidine, whereas in CRM 66, it is tyrosine.

show abstract

Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa.

Cited by 250 publications

References 31 publications

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

GPR107, a G-protein-coupled Receptor Essential for Intoxication by Pseudomonas aeruginosa Exotoxin A, Localizes to the Golgi and Is Cleaved by Furin

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1

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