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

Manal Abdel-Fattah,Hoda Mahmoud,Amira Embaby,Hesham Saeed,Farid Ataya,Manal Shalaby,Ahmed Hussein,Lamiaa El-Shennawy,Paulo Lee Ho

doi:10.1371/journal.pone.0213880

Abstract

The current study highlights, for the first time, cloning, overexpression and purification of the novel interferon epsilon (IFNƐ), from the Arabian camel Camelus dromedaries. The study then assesses the cytotoxicity of IFNε 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. dromedarius using 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 21.230 kDa. A BLAST search analysis revealed that the C. dromedarius IFNε shared high sequence identity with the IFN genes of other species, such as Camelus ferus, Vicugna pacos, and Homo sapiens. Expression of the camel IFNε cDNA in Escherichia coli gave a fusion protein band of 24.97 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.

Highlights

IFNs are members of a large cytokine family of evolutionarily conserved pleiotropic regulators of cellular functions; they are relatively low-molecular weight signaling proteins (20–25 kDa) usually glycosylated and produced by a variety of cells, such as epithelia, endothelia, stroma, and cells of the immune system [1,2,3]
Most information about type I IFNs has stemmed from the studies of IFNs from other species such as human, turkey, zebra fish, and bovine, but no published data is available on the Arabian camel IFNs [11, 20, 21]
The polymerase chain reaction (PCR) product corresponding to the 582 nucleotides represents the fulllength IFNε cDNA (Fig 1)

Summary

Introduction

IFNs are members of a large cytokine family of evolutionarily conserved pleiotropic regulators of cellular functions; they are relatively low-molecular weight signaling proteins (20–25 kDa) usually glycosylated and produced by a variety of cells, such as epithelia, endothelia, stroma, and cells of the immune system [1,2,3]. IFNs, induce a diverse range of biological functions and responses, including cell proliferation and differentiation, inflammation, chemotaxis, immune cell (natural killer cells and macrophages) activation, and apoptosis [5, 6]. Besides the autocrine activation of antiviral responses, type I IFNs function systematically to induce an antiviral state in the surrounding and distal cells [10, 11]. Hepatitis B, hepatitis C, and HIV are treated with IFN-α often in combination with other antiviral drugs [14, 15]

Objectives

Methods

Results