Recombinant production and affinity purification of the FraC pore forming toxin using hexa-His tag and pET expression cassette | ||
| Iranian Journal of Basic Medical Sciences | ||
| مقاله 5، دوره 20، شماره 4، تیر 2017، صفحه 380-385 اصل مقاله (1.1 M) | ||
| نوع مقاله: Original Article | ||
| شناسه دیجیتال (DOI): 10.22038/ijbms.2017.8577 | ||
| نویسندگان | ||
| Mehdi Imani* 1، 2؛ Hossein Zarei Jaliani3؛ Mohammad Hassan Kheirandish3؛ Mahnaz Azadpour3 | ||
| 1Department of Basic Sciences, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran | ||
| 2Department of Cellular and Molecular Biotechnology, Institute of Biotechnology, Urmia University, Urmia, Iran | ||
| 3Department of Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran | ||
| چکیده | ||
| 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. | ||
| کلیدواژهها | ||
| Fragaceatoxin C؛ Recombinant protein؛ Gene cloning؛ Hemolysis؛ Sea Anemone | ||
| مراجع | ||
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