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
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 Saeed1*, Manal Abdel-Fattah1, Ahmad Eldoksh1, Farid S. Ataya2 and Manal Shalaby3
ABSTRACT
Theopen 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 purified using on Ni-NTA Agarose affinity 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.
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August 2021
Vol. 53, Iss. 4, Pages 1201-1601
