Optimization of human interferon beta protein expression in Chinese hamster ovary cells

Background and aims: Human interferon beta-۱a (hIFNβ-۱a) is a ۲۲.۵-kDa glycoprotein used to treat diseases such as multiple sclerosis(MS). Because of appropriate post-translation modifications, protein isolation, and lack of toxicity in Chinese hamster ovary (CHO) cells,we cloned hIFNβ-۱a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinantprotein.Methods: The hIFNβ-۱a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and thensyntactically constructed in the pUC۵۷ vector. After confirmation, the synthesized sequence was cloned into the pcDNA۳.۱ expressionvector by using EcoRI and XhoI sites via Escherichia coli DH۵α competent cells. Then, the recombinant vector pcDNA-hHIFNβ۱a waslinearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened bygentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinantprotein expression was qualitatively evaluated using different techniques.Results: The hIFNβ۱a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ۱apcDNA۳.۱in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfectedCHO cells was performed using gentamicin. The protein expression was confirmed by RT-PCR, MTT assay, SDS-PAGE, and Western blot.Comparison of the optimized and control samples demonstrated that chemical treatment enhanced the protein expression.Conclusion: We achieved the stable clones of CHO cells expressing the active form of human interferon beta.

کلیدواژه ها:

hIFNβ-۱a ، CHO cells ، pcDNA۳.۱ expression vector ، lipofectamine ، MTT assay