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Vol. 64 No. 1: Issue 1

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Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli

https://doi.org/10.14715/cmb/2018.64.1.12
Hina Qaiser
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Farheen Aslam
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Saima Iftikhar
School of Biological Sciences, University of Punjab, Lahore, Pakistan
Adeen Farooq
Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan

Corresponding Author(s) : Hina Qaiser

hinaqaiser@lgu.edu.pk

Cellular and Molecular Biology, Vol. 64 No. 1: Issue 1

Abstract

Pseudomonas aeruginosa exotoxin A (PE) is a bacterial toxin composed of three domains namely: cell binding, translocation and enzymatic domain. The cytotoxic activity of PE is attributed to the enzymatic domain, which inhibits protein synthesis through ADP-ribosylation of EF-2. PE can be genetically modified to fight cancer. In this regard, a truncated and modified form of PE was produced that could be used for more potent immunotoxins. This modified form termed PE38KDEL was completely devoid of cell binding domain and parts of translocation domain II and Ib which are reported to be inessential for cytotoxicity of the toxin. The resultant expressed protein consisted of the essential translocation domain II and catalytic subunit (domain Ib, III). The deletions in the exotoxin A gene for truncated protein production were made via overlapping PCR extension. The amplicon was cloned in pTZ57r-T vector for DNA works and sub cloned in pET22b expression vector.  It is demonstrated here that PE38KDEL can be expressed in huge quantities in Escherichia coli by using the recombinant vector PE38KDEL/pET under control of T7 promoter and E. coli host strain BL21 (DE3) CodonPlus. The protein expression was optimized at 0.5 mM IPTG concentration for induction as soon as the OD600 nm reached 0.6 with 6 hours of post induction culturing at 37°C.  The recombinant protein was expressed both as soluble and inclusion body forms however the expression of the soluble form was more pronounced.

Keywords

Pseudomonas aeruginosa exotoxin A Cytotoxic immunotoxin PE38KDEL Expression.
Qaiser, H., Aslam, F., Iftikhar, S., & Farooq, A. (2018). Construction and recombinant expression of Pseudomonas aeruginosa truncated exotoxin A in Escherichia coli. Cellular and Molecular Biology, 64(1), 64–69. https://doi.org/10.14715/cmb/2018.64.1.12
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References
  1. Tarver T. Cancer Facts & Figures 2012. American Cancer Society (ACS) Atlanta, GA: American Cancer Society, 2012. J Consum Health Internet 2012; 16(3):366-367.
  2. Siegel RL, Miller KD and Jemal A. Cancer statistics, 2016. CA Cancer J Clin 2016; 66(1):7-30.
  3. Holohan C, Van Schaeybroeck S, Longley DB and Johnston PG. Cancer drug resistance: an evolving paradigm. Nat Rev Can 2013; 13(10):714.
  4. Li Z, Yu T, Zhao P and Ma J. Immunotoxins and cancer therapy. Cell Mol Immunol 2005; 2(2):106-112.
  5. Kreitman RJ. Immunotoxins for targeted cancer therapy. AAPS J 2006; 8(3):532-551.
  6. Aruna G. Immunotoxins: A review of their use in cancer treatment. J. Stem Cells Regen. Med. 2006; 1(1):31.
  7. Dosio F, Brusa P and Cattel L. Immunotoxins and anticancer drug conjugate assemblies: the role of the linkage between components. Toxins 2011; 3(7):848-883.
  8. Bernardes N, Chakrabarty AM and Fialho AM. Engineering of bacterial strains and their products for cancer therapy. Appl Microbio Biotech 2013; 97(12):5189-5199.
  9. Mathew M and Verma RS. Humanized immunotoxins: a new generation of immunotoxins for targeted cancer therapy. Cancer Sci 2009; 100(8):1359-1365.
  10. Engel JN. Molecular pathogenesis of acute Pseudomonas aeruginosa infections. In:Severe infections caused by Pseudomonas aeruginosa. (ed.), Springer US, 2003, pp.201-229
  11. Alewine C, Hassan R and Pastan I. Advances in anticancer immunotoxin therapy. The Oncologist 2015; 20(2):176-185.
  12. Allured VS, Collier RJ, Carroll SF and McKay DB. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc. Natl. Acad. Sci 1986; 83(5):1320-1324.
  13. Hwang J, Fitzgerald DJ, Adhya S and Pastan I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell 1987; 48(1):129-136.
  14. Jimenez PN, Koch G, Thompson JA, Xavier KB, Cool RH and Quax WJ. The multiple signaling systems regulating virulence in Pseudomonas aeruginosa. Microbiol Mol Bio Rev 2012; 76(1):46-65.
  15. Kreitman RJ and Pastan I. Accumulation of a recombinant immunotoxin in a tumor in vivo: fewer than 1000 molecules per cell are sufficient for complete responses. Cancer Res 1998; 58(5):968-975.
  16. Liu W, Onda M, Lee B, Kreitman RJ, Hassan R, Xiang L, et al. Recombinant immunotoxin engineered for low immunogenicity and antigenicity by identifying and silencing human B cell epitopes. Proc. Natl. Acad. Sci 2012; 109(29):11782-11787.
  17. Weldon JE, Xiang L, Chertov O, Margulies I, Kreitman RJ, FitzGerald DJ, et al. A protease resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity. Blood 2009; 113(16):3792-3800.
  18. Onda M, Beers R, Xiang L, Nagata S, Wang Q and Pastan I. An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes. Proc Nat Acad Sci 2008; 105(32):11311-11316.
  19. Baradaran B, Farajnia S, Majidi J, Omidi Y and Saeedi N. Recombinant Expression and Purification of Pseudomonas aeruginosa Truncated Exotoxin A in Escherichia coli. Pharma Sci 2013; 19(1):31.
  20. Chaudhary VK, FitzGerald DJ, Adhya S and Pastan I. Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin. Proc Nat Acad Sci 1987; 84(13):4538-4542.
  21. Kondo T, FitzGerald D, Chaudhary VK, Adhya S and Pastan I. Activity of immunotoxins constructed with modified Pseudomonas exotoxin A lacking the cell recognition domain. J Bio Chem 1988; 263(19):9470-9475.
  22. Theuer CP, Kreitman RJ, FitzGerald DJ and Pastan I. Immunotoxins made with a recombinant form of Pseudomonas exotoxin A that do not require proteolysis for activity. Cancer Res 1993; 53(2):340-347.
  23. Song S, Xue J, Fan K, Kou G, Zhou Q, Wang H, et al. Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli. Protein Expr Purif 2005; 44(1):52-57.
  24. Sambrook J and Russell DW. Purification of nucleic acids by extraction with phenol:chloroform. Cold Spring Harb Prot 2006; 2006(1):pdb. prot4455.
  25. Pastan I, Hassan R, FitzGerald DJ and Kreitman RJ. Immunotoxin treatment of cancer. Ann Rev Medicine 2007; 58(221-237.
  26. Saelinger C and Morris R. Intracellular trafficking of Pseudomonas exotoxin A. In:Basic Research and Clinical Aspects of Pseudomonas aeruginosa. (ed.), Karger Publishers, 1987, pp.149-159
  27. FitzGerald DJ, Willingham MC and Pastan I. Pseudomonas exotoxin-immunotoxins. In:Immunotoxins. (ed.), Springer, 1988, pp.161-173
  28. Collier J. Structure-activity relationships in diphtheria toxin and Pseudomonas aeruginosa exotoxin A. In:Immunotoxins. (ed.), Springer, 1988, pp.25-35
  29. Wick MJ, Frank D, Storey D and Iglewski B. Structure, function, and regulation of Pseudomonas aeruginosa exotoxin A. Annu Rev Microbiol 1990; 44(1):335-363.
  30. Mrsny RJ, Daugherty AL, McKee ML and FitzGerald DJ. Bacterial toxins as tools for mucosal vaccination. Drug Discov Today 2002; 7(4):247-258.
  31. Wolf P and Elsässer U. Pseudomonas exotoxin A: from virulence factor to anti-cancer agent. Int J Med Microbiol 2009; 299(3):161-176.
  32. Voulhoux R, Taupiac M-P, Czjzek M, Beaumelle B and Filloux A. Influence of deletions within domain II of exotoxin A on its extracellular secretion from Pseudomonas aeruginosa. J Bacteriol 2000; 182(14):4051-4058.
  33. Lv M, Qiu F, Li T, Sun Y, Zhang C, Zhu P, et al. Construction, Expression, and Characterization of a Recombinant Immunotoxin Targeting EpCAM. Mediators Inflamm 2015; 2015(
  34. Antignani A and FitzGerald D. Immunotoxins: the role of the toxin. Toxins 2013; 5(8):1486-1502.
  35. Seetharam S, Chaudhary VK, FitzGerald D and Pastan I. Increased cytotoxic activity of Pseudomonas exotoxin and two chimeric toxins ending in KDEL. J Biol Chem 1991; 266(26):17376-17381.
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Author Biographies

Hina Qaiser, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Lecturer, Department of Biology
Farheen Aslam, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Department of Biotechnology, Assistant Professor
Saima Iftikhar, School of Biological Sciences, University of Punjab, Lahore, Pakistan
School of Biological Sciences, Experimental Officer
Adeen Farooq, Department of Biotechnology, Lahore College for Women University, Lahore, Pakistan
Department of Biotechnology
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