The Arabian camel,Camelus dromedariusinterferon epsilon: functional expression,in vitrorefolding, purification and cytotoxicity on breast cancer cell lines MDA-MB-231 and MCF-7
AbstractThe current study highlights for the first time cloning, overexpression, purification, and assessing the cytotxcity of the novel interferon epsilon (IFNε), from the Arabian camelCamelus dromedarius, against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFNε) was isolated and cloned from the liver of the Arabian camel,C. dromedariususing reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFNε cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 22.953 kDa. A BLAST search analysis revealed that theC. dromedariusIFNε shared high sequence identity with the IFN genes of other species, such asCamelus ferus,Vicugna pacos, andHomo sapiens. Expression of the camel IFNε cDNA inEscherichia coligave a fusion protein band of 22.73 kDa after induction with either isopropyl β-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFNε protein was overexpressed in the form of inclusion bodies that were easily solubilized and refolded using SDS and KCl. The solubilized inclusion bodies were purified to apparent homogeneity using nickel affinity chromatography. We examined the effect of IFNε on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFNε inhibited cell survival in a dose dependent manner as observed by MTT assay, morphological changes and apoptosis assay. Caspase-3 expression level was found to be increased in MDA-MB-231 treated cells as compared to untreated cells.
