Online First Article The Arabian Camel, Camelus dromedarius Interferon Alpha: Cloning, Expression in Escherichia coli, in vitro Refolding and Cytotoxicity on Triple Negative Breast Cancer Cell Line MDA-MB-231 Hesham Saeed 1 *, Manal Abdel-Fattah 1 , Ahmad Eldoksh 1 , Farid S. Ataya 2 and Manal Shalaby 3 1 Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt. 2 Biochemistry Department, College of Science, Riyadh, King Saud University, Saudi Arabia. 3 Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientifc Research and Technology Applications, New Borg Al-Arab City, Alexandria, Egypt. Article Information Received 20 April 2020 Revised 30 May 2020 Accepted 02 June 2020 Available online 07 October 2020 Authors’ Contribution HS supervised the experiments and prepared the manuscript. MA con- ducted the experiments evaluated the results. AE evaluated and validated the results. FA supervied the study and did some experiments. MS helped in manuscript revision and preparation. Key words Camelus dromedaries, Cloning, Expression, Inclusion bodies, Interferon The open reading frame encoding interferon alpha (IFNα) of the camel liver, Camelus dromedarius was isolated and cloned using reverse transcription-PCR. Sequence analysis of that gene showed a 564- bp encoding a protein of 187 amino acids with a predicted molecular weight of 21 kDa. Basic local alignment search tool (BLAST) sequence analysis revealed that C. dromedarius IFNα gene shares high sequence identity with IFNα genes of other species, including C. ferus, Vicugna pacos, and Homo sapiens. Expression of C. dromedarius IFNα cDNA in Escherichia coli revealed a fusion protein with a weight of 22.5 kDa after induction of expression with IPTG for 5 h. The recombinant IFNα was expressed in the form of inclusion bodies that were separated and solubilized in vitro and the protein was refolded using SDS and KCl. The folded protein is then purifed using on Ni-NTA Agarose affnity chromatography and the purity was judged by SDS-PAGE. Moreover, the effect of the recombinant IFNα of the viability of cancer cell line was assessed by MTT assay. Morphological study showed that C. dromedarius IFNα protein inhibited cell survival of MDA-MB-231 triple negative breast cancer cells. INTRODUCTION T he term interferon (IFN) was frst coined by Alick Isaacs and Jean Lindemann in 1957 at the National Institute for Medical Research in London to describe an antiviral compound produced by virus infected chick cells that were able to interfere with viral infection (Isaacs and Lindemann, 1957). Since then, research pertaining to the discovery, characterization, and development of novel IFNs has continued for over 60 years (Meager, 2009). IFNs belong to a pleiotropic family of cytokines that play an important role in controlling cellular growth and apoptosis, and in the response to infections (Kaplan et al., 2017). IFNs are glycosylated proteins having molecular weight ranging from 20 to 25 kDa. They are produced in response to a variety stimuli including viral, bacterial, * Corresponding author: hsaeed1@ksu.edu.sa 0030-9923/2021/0001-0001 $ 9.00/0 Copyright 2021 Zoological Society of Pakistan parasitic infections, infammation, and tumorigenesis by various body cells like epithelia, endothelia, stroma, and cells of the immune system (Baldo, 2014; Borish and Steinke, 2003; Vacchelli et al., 2013; Peng et al., 2007). IFNs play important role in cell proliferation and differentiation, activation of immune cells, chemotaxis, infammation, and apoptosis (Tayal and Kalra, 2008; Vacchelli et al., 2012). IFNs are classifed-based on the receptors they interact with-into three major classes namely, type I, II, and III. Each type is encoded from different gene and has specifc chromosomal localization, protein structures and biological activity (Fischer et al., 2018). Type II and III IFNs consist only of IFNγ and IFN while type I IFN consists of IFNα, β, δ, ε, ζ, κ ,, and ω (Klotz et al., 2017). The most common cytokine that has the longest record of use in clinical oncology is Type I IFNα as it is used in over 40 countries for the treatment of hematological malignancies and certain solid tumors such as melanoma, renal carcinoma, and Kaposi’s sarcoma (Meager, 2009; Ferrantini et al., 2007). Moreover, ABSTRACT Pakistan J. Zool., pp 1-11, 2021. DOI: https://dx.doi.org/10.17582/journal.pjz/20200420000447