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 | [Pakistan Journal of Zoology • 2021]

Author(s):

1. Hesham Saeed: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University,Alexandria,Egypt

2. Manal Abdel-Fattah: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University,Alexandria,Egypt

3. Ahmad Eldoksh: Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University,Alexandria,Egypt

4. Farid S. Ataya: Biochemistry Department, College of Science, Riyadh, King Saud University, Saudi Arabia.

5. Manal Shalaby: Genetic Engineering and Biotechnology Research Institute (GEBRI), City for Scientific Research and Technology Applications, New Borg Al-Arab City,Alexandria,Egypt

Abstract:

The open reading frame encoding interferon alpha (IFNa) of the camel liver, Camelus dromedarius was isolated and cloned using reverse transcription-PCR. Sequence analysis of that gene showed a 564bp 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 IFNa gene shares high sequence identity with IFNa genes of other species, including C. ferus, Vicugna pacos, and Homo sapiens. Expression of C. dromedarius IFNa 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 IFNa 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 purified using on Ni-NTA Agarose affinity chromatography and the purity was judged by SDS-PAGE. Moreover, the effect of the recombinant IFNa of the viability of cancer cell line was assessed by MTT assay. Morphological study showed that C. dromedarius IFNa protein inhibited cell survival of MDA-MB-231 triple negative breast cancer cells.

Page(s): 1525-1535

DOI: 10.17582/journal.pjz/20200420000447

Published: Journal: Pakistan Journal of Zoology, Volume: 53, Issue: 4, Year: 2021

Keywords:

Interferon , Cloning , expression , Camelus dromedaries , Inclusion bodies

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