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<p>Exotoxin A is a key virulence factor of <i>Pseudomonas aeruginosa</i> that induces target cell death by inhibiting protein synthesis in eukaryotic cells, making it a potential candidate for cancer therapy. This study aimed to heterologously express Exotoxin A using the pET28a expression vector. Initially, the gene sequence of Exotoxin A, along with its toxicity and immunogenicity, was analyzed using bioinformatics tools. Subsequently, for heterologous protein expression, the Exotoxin A gene from <i>P. aeruginosa</i> (ATCC 27853) was amplified by polymerase chain reaction (PCR) and cloned into the pET28a expression vector. Exotoxin A (Exo-HB) was expressed in <i>E. coli</i> BL21 in the presence of 1&#xa0;mM IPTG as an inducer. The protein was purified using a nickel affinity chromatography column with an imidazole gradient from 10 to 100&#xa0;mM. Moreover, native production of Exotoxin A (ExoHBN) was conducted from <i>P. aeruginosa</i>. The cytotoxicity of Exotoxin A was assessed on the A549 cell line and <i>Saccharomyces cerevisiae</i>. The heterologously produced protein Exo-HB was purified to 100&#xa0;mM. Cytotoxicity tests demonstrated that Exo-HB and ExoHBN exhibited maximum lethality on A549 cells at concentrations of 1.8&#xa0;μg/mL and 2.8&#xa0;μg/mL, respectively, within 24&#xa0;h. Additionally, treatment of yeast cells with Exo-HB showed that a concentration of 13.5&#xa0;μg/mL reduced yeast colony numbers by 57%. This study is the first to investigate the effects of Exotoxin A on yeast. The results contribute to a better understanding of the mechanisms behind Exotoxin A toxicity in eukaryotic organisms and lay the groundwork for future research in this area.</p>

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Heterologous expression of Pseudomonas aeruginosa Exotoxin A and evaluation of its functional activity

  • 期刊论文

  • 发表日期：

    2026年7月1日

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  • Hanieh Banaeyoun,
  • Shamsozoha Abolmaali,
  • Shakiba Darvish Alipour Astaneh

摘要

Exotoxin A is a key virulence factor of Pseudomonas aeruginosa that induces target cell death by inhibiting protein synthesis in eukaryotic cells, making it a potential candidate for cancer therapy. This study aimed to heterologously express Exotoxin A using the pET28a expression vector. Initially, the gene sequence of Exotoxin A, along with its toxicity and immunogenicity, was analyzed using bioinformatics tools. Subsequently, for heterologous protein expression, the Exotoxin A gene from P. aeruginosa (ATCC 27853) was amplified by polymerase chain reaction (PCR) and cloned into the pET28a expression vector. Exotoxin A (Exo-HB) was expressed in E. coli BL21 in the presence of 1 mM IPTG as an inducer. The protein was purified using a nickel affinity chromatography column with an imidazole gradient from 10 to 100 mM. Moreover, native production of Exotoxin A (ExoHBN) was conducted from P. aeruginosa. The cytotoxicity of Exotoxin A was assessed on the A549 cell line and Saccharomyces cerevisiae. The heterologously produced protein Exo-HB was purified to 100 mM. Cytotoxicity tests demonstrated that Exo-HB and ExoHBN exhibited maximum lethality on A549 cells at concentrations of 1.8 μg/mL and 2.8 μg/mL, respectively, within 24 h. Additionally, treatment of yeast cells with Exo-HB showed that a concentration of 13.5 μg/mL reduced yeast colony numbers by 57%. This study is the first to investigate the effects of Exotoxin A on yeast. The results contribute to a better understanding of the mechanisms behind Exotoxin A toxicity in eukaryotic organisms and lay the groundwork for future research in this area.