Recombinant production and affinity purification of the FraC pore forming toxin using hexa-His tag and pET expression cassette

Document Type: Original Article

Authors

1 Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran

2 Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran

3 Department of Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Abstract

Objective(s): A newly-introduced protein toxin from a sea anemone, namely fragaceatoxin C is a protein with molecular weight of 20 kDa and pore-forming capability against cell membranes has recently grasped great attentions for its function. In this study, its coding sequence cloned as a fusion protein with His-tag for simple production and rapid purification.
Materials and Methods: After PCR amplification using NcoI and HindIII-harboring primers, the gene fragment was cloned into pET-28a(+). Escherichia coli BL21 was used for expression of constructed vector and toxin expression was verified by SDS-PAGE. For one-step purification Ni-NTA sepharose  affinity chromatography was employed. For examination of purified toxin function, RBC hemolytic test was conducted.
Results: The results showed that the FraC-coding gene was successfully cloned between NcoI and HindIII restriction sites and purified with affinity chromatography. Densitometric analysis represented the purity of approximately 97%. Hemolytic test indicated the purified FraC had remarkable lytic activity on RBC and almost lysed 50% of cells at the concentration value of 6.25 nM.
Conclusion: The results indicated that not only purified toxin preserved its activity during expression and purification processes but also exerted its function at lower concentrations so that even the 0.09 nM displayed hemolytic effect.

Keywords


1. Anderluh G, Maček P. Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria). Toxicon 2002; 40:111-124.

2. Honma T, Shiomi K. Peptide toxins in sea anemones: structural and functional aspects. Mar Biotechnol 2006; 8:1-10.

3. Soletti RC, de Faria GP, Vernal J, Terenzi H, Anderluh G, Borges HL, et al. Potentiation of anticancer-drug cytotoxicity by sea anemone pore-forming proteins in human glioblastoma cells. Anti-Cancer Drugs 2008; 19:517-525 510.

4. Bellomio A, Morante K, Barlič A, Gutiérrez-Aguirre I, Viguera AR, González-Mañas JM. Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea. Toxicon 2009; 54:869-880.

5. Mechaly AE, Bellomio A, Gil-Cartón D, Morante K, Valle M, González-Mañas JM, et al. Structural Insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure 2011; 19:181-191.

6. Wang Y, Chua KL, Khoo HE. A new cytolysin from the sea anemone, Heteractis magnifica: isolation, cDNA cloning and functional expression. Biochim Biophys Acta  2000; 1478:9-18.

7. Anderluh G, Barlič A, Podlesek Z, Maček P, Pungerčar J, Gubenšek F, et al. Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone. Eur J Biochem 1999; 263:128-136.

8. Avila AD, de Acosta CM, Lage A. A new immunotoxin built by linking a hemolytic toxin to a monoclonal antibody specific for immature T lymphocytes. Int J Cancer 1988; 42:568-571.

9. Pederzolli C, Belmonte G, Serra MD, Macek P, Menestrina G. Biochemical and cytotoxic properties of conjugates of transferrin with equinatoxin II, a cytolysin from a sea anemone. Bioconjugate Chem 1995; 6:166-173.

10. Panchal RG, Cusack E, Cheley S, Bayley H. Tumor protease-activated, pore-forming toxins from a combinatorial library. Nat Biotech 1996; 14:852-856.

11. Patyar S, Joshi R, Byrav D, Prakash A, Medhi B, Das B. Review Bacteria in cancer therapy: a novel
experimental strategy. J Biomed Sci 2010; 17:21-30.

12. Michl P, Gress T. Bacteria and bacterial toxins as therapeutic agents for solid tumors. Curr Cancer Drug Targets 2004; 4:689-702.

13. Potrich C, Tomazzolli R, Dalla Serra M, Anderluh G, Malovrh P, Maček P, et al. Cytotoxic activity of a tumor protease-activated pore-forming toxin. Bioconjugate Chemistry 2005; 16:369-376.

14.Thorpe J, Sole-CAVA AM. Population structuring, gene dispersal and reproduction in the Actinia equina species group. Oceanogr Mar Biol 1999; 37:129.

15.Tanaka K, Caaveiro JM, Morante K, González-Mañas JM, Tsumoto K. Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat Commun 2015; 6.

16.Yang WS, Park SO, Yoon A-R, Yoo JY, Kim MK, Yun C-O, et al. Suicide cancer gene therapy using pore-forming toxin, streptolysin O. Mol Cancer Ther 2006; 5:1610-1619.

17. Soletti RC, de Faria GP, Vernal J, Terenzi H, Anderluh G, Borges HL, et al. Potentiation of anticancer-drug cytotoxicity by sea anemone pore-forming proteins in human glioblastoma cells. Anticancer Drugs 2008; 19:517-525.

18. Parker MW, Feil SC. Pore-forming protein toxins: from structure to function. Prog Biophys Mol Biol 2005; 88:91-142.

19. Koths K. Recombinant proteins for medical use: the attractions and challenges. Curr Opin Biotechnol 1995; 6:681-687.

20. Parenti G, Andria G, Ballabio A. Lysosomal storage diseases: from pathophysiology to therapy. Ann Rev Med 2015; 66:471-486.

21. Martino MM, Brkic S, Bovo E, Burger M, Schaefer DJ, Wolff T, et al. Extracellular matrix and growth factor engineering for controlled angiogenesis in regenerative medicine. Front Bioeng Biotechnol 2015; 3.

22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72:248-254.

23. Anderluh G, Križaj I, Štrukelj B, Gubenšek F, Maček P, Pungerčar J. Equinatoxins, pore-forming proteins from the sea anemone Actinia equina, belong to a multigene family. Toxicon 1999; 37:1391-1401.