Expression, purification, and cytotoxic evaluation of IL24-BR2 fusion protein

Marjan Pourhadi , Fahimeh Jamalzade, Ali Jahanian-Najafabadi, Fatemeh Shafiee

Abstract


Interleukin (IL) 24 is a pro-inflammatory and tumor suppressor cytokine capable of inducing selective apoptosis in various cancer cells. BR2, on the other hand, is an anti-microbial peptide with selective penetrability to the cancer cells. In this study, we aimed to produce and purify a fusion protein containing IL24 as the toxic moiety fused to BR2, as targeting moiety, and then to evaluate its cytotoxic activities. For this purpose, the coding sequence of IL24-BR2 fusion protein and IL24 were cloned into the pET28a vector and used to transform E. coli BL21 (DE3) cells. Following induction of expression, protein purification performed using Ni-NTA chromatography. SDS-PAGE and western blotting were performed to confirm the expression and purification. Finally, cytotoxic effects of the purified proteins were evaluated on MCF-7 and HUVEC cell lines. Analysis of crude lysate of induced recombinant E. coli BL21 (DE3) bacteria and also purified proteins showed a band of approximately 22 and 18 KDa on SDS-PAGE and western blotting for IL24-BR2 and IL24, respectively. Finally, statistical analysis showed significant cytotoxic effects of IL24-BR2 on MCF-7 cells at 10, 20, and 40 µg/mL concentrations compared to IL24 alone, which showed no significant cytotoxic effects on cancer cells except in the highest concentration.  In conclusion, production and purification of IL24-BR2 fusion protein with potential specific toxicity toward cancer cells was successfully achieved. However, further investigation of the cytotoxic effects of this fusion protein on other cell lines and in vivo cancer models must be performed.


Keywords


BR2; Cytotoxicity; Fusion protein; IL24; IL24-BR2.

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