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J Shahrekord Univ Med Sci. 2021;23(2): 68-75.
doi: 10.34172/jsums.2021.11
  Abstract View: 92
  PDF Download: 99

Original Article

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

Nasrin Xodadadi 1 ORCID logo, Alireza Saeidinia 1* ORCID logo, Mehdi Zeinoddini 1 ORCID logo, Rasoul Khalilzadeh 1 ORCID logo

1 Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran
*Corresponding Author: Alireza Saeidinia, Faculty of Chemistry and Chemical Engineering, Malek Ashtar University of Technology, Tehran, Iran. Postcode: 1774-15875. Telephone: +982122945141, Fax: +982122935341, Email: ar_saeedi@ mut.ac.ir

Abstract

Background and aims: Human interferon beta-1a (hIFNβ-1a) is a 22.5-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-1a encoding sequence into these cells by recombinant DNA technology to achieve stable expression of this recombinant protein.

Methods: The hIFNβ-1a encoding sequence was designed based on the CHO cells’ codon usage and the Gene Bank data, and then syntactically constructed in the pUC57 vector. After confirmation, the synthesized sequence was cloned into the pcDNA3.1 expression vector by using EcoRI and XhoI sites via Escherichia coli DH5α competent cells. Then, the recombinant vector pcDNA-hHIFNβ1a was linearized by BglII and transfected into the CHO cells using lipofectamine. The transfected cells were proliferated and screened by gentamicin. Certain concentrations of zinc sulfate, DMSO, and glycerol were used to enhance protein expression. Finally, the recombinant protein expression was qualitatively evaluated using different techniques.

Results: The hIFNβ1a integrity was confirmed by DNA sequencing and specific software. The construction and sub-cloning of hIFNβ1a-pcDNA3.1 in E. coli were confirmed by colony-PCR with specific primers and restriction enzyme mapping. The screening of transfected CHO 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.

Keywords: hIFNβ-1a, CHO cells, pcDNA3.1 expression vector, lipofectamine, MTT assay
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Submitted: 16 Sep 2019
Accepted: 20 Sep 2020
ePublished: 30 Jun 2021
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