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


  1. Allured VS, Collier RJ, Carroll SF, McKay DB. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci U S A 1986; 83(5): 1320-4.
  2. Hwang J, Fitzgerald DJ, Adhya S, Pastan I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 1987; 48(1): 129-36.
  3. Chaudhary VK, Xu YH, FitzGerald D, Adhya S, Pastan I. Role of domain II of Pseudomonas exotoxin in the secretion of proteins into the periplasm and medium by Escherichia coli. Proc Natl Acad Sci U S A 1988; 85(9): 2939-43.
  4. Kreitman RJ, Pastan I. Importance of the glutamate residue of KDEL in increasing the cytotoxicity of Pseudomonas exotoxin derivatives and for increased binding to the KDEL receptor. Biochem J 1995; 307( Pt 1): 29-37.
  5. Sambrook J. Molecular Cloning: A Laboratory Manual. 2nd ed. New York, NY: Cold Spring Harbor Laboratory Press; 1989.
  6. Pai LH, Wittes R, Setser A, Willingham MC, Pastan I. Treatment of advanced solid tumors with immunotoxin LMB-1: an antibody linked to Pseudomonas exotoxin. Nat Med 1996; 2(3): 350-3.
  7. Pastan I. Immunotoxins containing Pseudomonas exotoxin A: a short history. Cancer Immunol Immunother 2003; 52(5): 338-41.
  8. Kreitman RJ, Wilson WH, White JD, Stetler-Stevenson M, Jaffe ES, Giardina S, et al. Phase I trial of recombinant immunotoxin anti-Tac(Fv)-PE38 (LMB-2) in patients with hematologic malignancies. J Clin Oncol 2000; 18(8): 1622-36.
  9. Kreitman RJ, Wilson WH, Bergeron K, Raggio M, Stetler-Stevenson M, Fitzgerald DJ, et al. Efficacy of the anti-CD22 recombinant immunotoxin BL22 in chemotherapy-resistant hairy-cell leukemia. N Engl J Med 2001; 345(4): 241-7.
  10. Frankel AE, Neville DM, Bugge TA, Kreitman RJ, Leppla SH. Immunotoxin therapy of hematologic malignancies. Semin Oncol 2003; 30(4): 545-57.
  11. Siegall CB, Liggitt D, Chace D, Tepper MA, Fell HP. Prevention of immunotoxin-mediated vascular leak syndrome in rats with retention of antitumor activity. Proc Natl Acad Sci U S A 1994; 91(20): 9514-8.
  12. Baluna R, Vitetta ES. An in vivo model to study immunotoxin-induced vascular leak in human tissue. J Immunother 1999; 22(1): 41-7.
  13. Baluna R, Rizo J, Gordon BE, Ghetie V, Vitetta ES. Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome. Proc Natl Acad Sci U S A 1999; 96(7): 3957-62.
  14. Smallshaw JE, Ghetie V, Rizo J, Fulmer JR, Trahan LL, Ghetie MA, et al. Genetic engineering of an immunotoxin to eliminate pulmonary vascular leak in mice. Nat Biotechnol 2003; 21(4): 387-91.
  15. Siegall CB, Liggitt D, Chace D, Mixan B, Sugai J, Davidson T, et al. Characterization of vascular leak syndrome induced by the toxin component of Pseudomonas exotoxin-based immunotoxins and its potential inhibition with nonsteroidal anti-inflammatory drugs. Clin Cancer Res 1997; 3(3): 339-45.