The Arabian camel, Camelus dromedarius interferon epsilon: Functional expression, in vitro refolding, purification and cytotoxicity on breast cancer cell lines.

ABSTRACT: The current study highlights, for the first time, cloning, overexpression and purification of the novel interferon epsilon (IFN?), from the Arabian camel Camelus dromedaries. The study then assesses the cytotoxicity of IFN? against two human breast cancer cell lines MDA-MB-231 and MCF-7. Full-length cDNA encoding interferon epsilon (IFN?) was isolated and cloned from the liver of the Arabian camel, C. dromedarius using reverse transcription-polymerase chain reaction. The sequence analysis of the camel IFN? cDNA showed a 582-bp open reading frame encoding a protein of 193 amino acids with an estimated molecular weight of 21.230 kDa. A BLAST search analysis revealed that the C. dromedarius IFN? shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFN? cDNA in Escherichia coli gave a fusion protein band of 24.97 kDa after induction with either isopropyl ?-D-1-thiogalactopyranoside or lactose for 5 h. Recombinant IFN? 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 IFN? on two breast cancer cell lines MDA-MB-231 and MCF-7. In both cell lines, IFN? 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.

SUBMITTER: Abdel-Fattah M 

PROVIDER: S-EPMC6730848 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature