Cloning, Expression and Evaluation of Pseudomonas Aeruginosa Exotoxin A

Document Type : Original Article (s)

Authors

1 PhD Candidate, Department of Molecular Medicine, Biotechnology Research Centre, Pasteur Institute of Iran, Tehran, Iran

2 Assistant Professor, Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran

3 Assistant Professor, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

4 Assistant Professor, Department of Molecular Medicine, Pasteur Institute of Iran, Amol, Iran

5 PhD Candidate, Department of Molecular Medicine, Pasteur Institute of Iran, Amol, Iran

6 Pasteur Institute of Iran, North Research Center, Amol, Iran

7 Assistant Professor, Department of Rabies, Pasteur Institute of Iran, Tehran, Iran

Abstract

Background: Nowadays, in many studies related to the treatment of various cancers, toxic compounds are targeted against cancer cells. One of the most effective compounds is Pseudomonas exotoxin A. The purpose of this study was to investigate the expression, purification, and in-vitro evaluation of a short form of the toxin in a prokaryotic expression system.Methods: The short form of the toxin (PE38) was amplified via polymerase chain reaction (PCR) using primers containing HindIII and NdeI restriction enzyme sites from pUC57-PE38. The polymerase chain reaction product was digested and subcloned in the pET-26b expression vector. The expression vector was separately transformed into the BL21(DE3), BL21(DE3) plys S and Rosetta Escherichia coli strains. Recombinant bacteria were cultured and induced and the resulted PE38 protein purified using metal affinity column chromatography. The toxicity effect of PE38 protein was assessed on HUVEC and 293KDR eukaryotic cells.Findings: The gene was successfully cloned into the expression vector and the accuracy of construct was confirmed via restriction map analysis and sequencing. The expression was found more in BL21(DE3) than the other strains. The toxicity effect was observed at the same level for both HUVEC and 293KDR cells.Conclusion: The lethal dose of truncated toxin is more than the previous study (1000-fold), where the targeted vascular endothelial growth factor (VEGF121) was fused to the truncated toxin. 

Keywords

References

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Journal of Isfahan Medical School
Volume 32, Issue 294 - Serial Number 294
May and June 2014
Pages 1110-1118

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History

  • Receive Date: 09 September 2013
  • Revise Date: 02 November 2024
  • Accept Date: 06 April 2022

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