Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Sep 6;14(9):e0213880.
doi: 10.1371/journal.pone.0213880. eCollection 2019.

The Arabian Camel, Camelus Dromedarius Interferon Epsilon: Functional Expression, in Vitro Refolding, Purification and Cytotoxicity on Breast Cancer Cell Lines

Affiliations
Free PMC article

The Arabian Camel, Camelus Dromedarius Interferon Epsilon: Functional Expression, in Vitro Refolding, Purification and Cytotoxicity on Breast Cancer Cell Lines

Manal Abdel-Fattah et al. PLoS One. .
Free PMC article

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Agarose gel (1.5%) electrophoresis of PCR product for C. dromedarius interferon epsilon gene (Lane 2).
Lane 1 represents 100 base pair DNA ladder.
Fig 2
Fig 2. Alignment of the deduced amino acid sequence of C. dromedrius IFNε with IFNε from other species.
Fig 3
Fig 3. Phylogenetic relationship of C. dromedarius interferon epsilon and sequences from other species.
Maximum likelihood tree based on complete coding sequences deposited in GenBank. Values at nodes are bootstrap ≥50%, obtained from 1000 re-samplings of the data.
Fig 4
Fig 4. Nucleotide and deduced amino acid encoding region of C. dromedarius IFNε.
Important amino acid residues and regions include: residues contact to N-Acetyl-2-Deoxy- are in box; residues contact to SO4 ion are in bold underline; residue contact to Zn +2 are bold double underline, conserved amino acid residues in IFNε protein is in bold dashed underline, residues involved in IFNAR-1 binding are in circle and residues involved in IFNAR-2 binding are in bold dashed box. Arrows indicates the location of the forward and reverse primers with restriction enzyme sites are in bold underline italics.
Fig 5
Fig 5
(a) Sequence annotations for C. dromedarius IFNε showing the location of α-helices and residues contact to ligand and ions. Secondary structure by homology active sites residues from PDB site record (); residues contacts to ligand (*) and to ions (*). (b) Predicted 3D structure of C. dromedarius IFNε protein shows the overall secondary structure in cartoon form; ribbon form (c) and DNA binding form (d). Components of secondary structure are α-helices (blue), coils (green) and turns (red). Alpha helices are labelled from A to F. (e) Model-template alignment of amino acid residues of C. dromedarius IFNε and H. sapiens IFNα2. Components of the secondary structure are shown in blue (α-helices) and brown (coils). Identical amino acid residues are in bold black. (f) Predicted 3D structure model of C. dromedarius based on this model template alignment.
Fig 6
Fig 6
(a) SDS-PAGE (12%) for un-induced E. coli DE3 (BL21) pLysS pET28-a (+) harboring C. dromedarius IFNε cDNA (Lanes 2 and 3) and lactose induced culture (Lanes 4–7). (b) SDS-PAGE (12%) for un-induced E. coli DE3 (BL21) pLysS pET28-a (+) harboring C. dromedarius IFNε cDNA (Lane 2), IPTG induced culture supernatant (Lane 3), IPTG induced culture inclusion bodies (Lane 4), lactose induced culture supernatant (Lane 5) and lactose induced inclusion bodies (Lane 6). Lane 1 represents pre-stained protein molecular weight markers. Induction was carried out for 5 h at 1 mM IPTG and 2 g/L lactose in the fermentation medium. Arrow indicates the location of inclusion bodies.
Fig 7
Fig 7
(a) Transmission electron microscope micrograph for normal E. coli BL21 (DE3) pLysS harboring pET28a (+) carrying C. dromedarius IFNε gene becomes to form inclusion bodies, dark spots when induced to overexpress the recombinant protein. Direct magnification was 10,000 x. (b), (c) and (d) Scanning electron micrograph for the inclusion body showing a spherical particle of a diameter ranging from 0.5 to 1.0 μm. Direct magnification was 35,000 x for b and c and 50,000 x for d.
Fig 8
Fig 8
(a) SDS-PAGE of C. dromedarius IFNε inclusion bodies (Lane 2) and solubilized inclusion bodies (Lane 3). (b) Elution profile of C. dromedarius recombinant IFNε after nickel affinity chromatography. Column flow rate was adjusted to be 3 mL/5 min. Arrow indicates the fraction at which buffer was changed to contain imidazole at a concentration of 500 mM as eluent. (c) SDS-PAGE (12%) electrophoresis of nickel affinity purified refolded C. dromedarius IFNε, fraction # 21 (Lane 2). (d) SDS-PAGE (12%) for nickel affinity purified recombinant C. dromedarius IFNε (Lanes 2–4, 5–15 μg purified protein was loaded into each well). Lane 1 represents pre-stained protein molecular weight markers.
Fig 9
Fig 9
(a) Recombinant Arabian camel IFNε alters the morphology of breast cancer cell lines MDA-MB-231 (upper) and MCF-7 (lower). (b) Interferon epsilon inhibits the survival of breast cancer cells. Cells were treated with different concentrations of IFNƐ for 48 h. MTT assay was performed and percentage cell viability was calculated compared to control cells. GraphPad Prism 6 was used to calculate the IC50 of IFNƐ: 5.65±0.2 μM and 3.91±0.6 μM for MDA-MB-231 and MCF-7 cells respectively. Experiments were repeated at least 3 times in triplicate. (c) Interferon epsilon induces apoptosis in breast cancer cells. Cells were treated with 5 μM IFNε protein for 48 h. Apoptosis assay was performed, and the percentage cell viability was calculated (*p<0.5, **p<0.1 and ***p<0.01). (d) Expression of caspase-3 in MDA-MB-231 cell line untreated and recombinant IFNε treated cells at a concentration of 3 and 6 μM.

Similar articles

See all similar articles

References

    1. Baldo BA. Side effects of cytokines approved for therapy. Drug Saf. 2014; 37: 921–943. 10.1007/s40264-014-0226-z - DOI - PubMed
    1. Borish LC, Steinke JW. Cytokines and chemokines. J Allergy Clin Immunol. 2003; 111: S460–S475. 10.1067/mai.2003.108 - DOI - PubMed
    1. Vacchelli E, Eggermont A, Fridman WH, Galon J, Zitvogel L, Kroemer G, et al. Trial Watch Immunostimulatory cytokines. Oncoimmunology. 2013; 2(7): E24850 10.4161/onci.24850 - DOI - PMC - PubMed
    1. Peng FW, Duan ZJ, Zheng LS, Xie ZP, Gao HC, Zhang H, et al. Purification of recombinant human interferon-epsilon and oligonucleotide microarray analysis of interferon-epsilon-regulated genes. Protein Expr Purif. 2007; 53: 356–362. 10.1016/j.pep.2006.12.013 - DOI - PubMed
    1. Tayal V, Kalra BS. Cytokines and anti-cytokines as therapeutics—an update. Trial Watch: Immunostimulatory cytokines. Eur J Pharmacol. 2008; 579: 1–12. 10.1016/j.ejphar.2007.10.049 - DOI - PubMed

Grant support

The authors received no specific funding for this work.
Feedback