THE EXPRESSION AND PURIFICATION OF OCTA-ARGININE APOPTIN AND ITS ABILITY TO KILL CANCER CELLS
DOI:
https://doi.org/10.22159/ijpps.2016v8i10.11455Keywords:
Recombinant apoptin, Escherichia coli, Anti- cancerAbstract
Objective: In this research, chicken anemia virus apoptin optimized genetically for expression in Escherichia coli and also modified using (His)6 tag, (Arg)8 tag, and HlyA tag intended for purification needs, penetration enhancement, and secretion from bacterial host to the growth media.
Methods: The modified apoptin gene was optimized using an Integrated DNA Technology (IDT). The gene (606 bp) then ordered and synthesized by Eurofins. The apoptin gene was expressed using E. coli BL21 CodonPlus as host, in cultivation temperature of 37 °C, and 25 °C and purified using Ni-NTA agarose beads. The addition of (His) 6 tag enabled the apoptin to be purified in only one step by using nickel column. The expression and purification data analyzed qualitatively as well as quantitatively using SDS-PAGE. MTT assay was used to identify the antitumor effect of octa arginine-apoptin to two kinds of cancer cells, cervix HeLa cancer cell and colon Widr cancer cell. The viability of cell was analyzed when the cell incubated in the variation concentration protein for 72 h.
Results: The constructed apoptin gene were expressed in E. coli successfully. The MTT assay indicated that Octaarginin-Apoptin was able to induce apoptosis of HeLa and Widr cells lines in a dose-dependent manner. The recombinant apoptin without tagging with octa-arginine, have no ability to induce apoptosis of HeLa and Widr cells lines.
Conclusion: This octa arginine-apoptin may in the future allow the development of a therapeutic protein that is able to kill cancer cells specifically.
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