Design, construction, and expression of recombinant human interferon beta gene in CHO-s cell line using EBV-based expression system

Mohadeseh Shayesteh , Fahimeh Ghasemi, Fatemeh Tabandeh , Bagher Yakhchali, Mehdi Shakibaie


Background and purpose: Codon optimization has been considered as a powerful strategy to increase the expression level of protein therapeutics in mammalian cells. As an empirical approach to study the effects of the codon usage and GC content on heterologous gene expression in suspension adapted Chinese hamster ovary (CHO-s) cells, we redesigned the recombinant human interferon beta (rhIFN-β) gene based on the codon preference of the CHO cell in a way to increase the GC content in the third position of each codon.

Experimental approach: The nucleotide sequence of the codon-optimized rhIFN-β was synthesized in parallel with the wild-type and expressed transiently in CHO-s cells using Epstein-Bar virus (EBV)-based expression system. The protein expression of the rhIFN-β by codon-optimized and wild-type genes were quantified using ELISA test.

Findings / Results: The results indicated a 2.8-fold increase in the expression level of the biologically active form of the rhIFN-β by codon-optimized sequence.

Conclusion and implications: These results shed light on the capability of codon optimization to create a stable CHO cell for scaling up the production of recombinant therapeutics such as rhIFN-β.




Codon optimization; CHO-s cells; EBV-based expression system; Human interferon beta; Recombinant protein production.

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González-Navajas JM, Lee J, David M, Raz E. Immunomodulatory functions of type I interferons. Nat Rev Immunol, 2012;12(2):125-135.

DOI: 10.1038/nri3133.

Gibbert K, Schlaak JF, Yang D, Dittmer U. IFN‐α subtypes: distinct biological activities in anti‐viral therapy. Br J Pharmacol. 2013;168(5):1048-1058. DOI: 10.1111/bph.12010.

Villela Dr A, Renard G, Palma MS, Chies JM, Dalmora SL, Basso LA, et al. Human interferon B1 ser17: coding DNA synthesis, expression, purification and characterization of bioactive recombinant protein. J Microb Biochem Technol. 2010;2(5):111-117.

DOI: 10.4172/1948-5948.1000034.

Rodriguez J, Spearman M, Tharmalingam T, Sunley K, Lodewyks C, Huzel N, et al. High productivity of human recombinant beta-interferon from a low-temperature perfusion culture. J Biotechnol. 2010;150(4):509-518.

DOI: 10.1016/j.jbiotec.2010.09.959.

Runkel L, Meier W, Pepinsky RB, Karpusas M, Whitty A, Kimball K, et al. Structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (IFN-beta). Pharm Res. 1998;15(4):641-649.

DOI: 10.1023/a:1011974512425.

Meager A, Gaines Das R. Biological standardization of human interferon beta: establishment of a replacement world health organization international biological standard for human glycosylated interferon beta. J Immunol Methods. 2005;306(1-2):1-15.

DOI: 10.1016/j.jim.2005.08.007.

Song K, Yoon IS, Kim NA, Kim DH, Lee J, Lee HJ, et al. Glycoengineering of interferon-β 1a improves its biophysical and pharmacokinetic properties. PLoS One. 2014;9(5):e96967,1-14.

DOI: 10.1371/journal.pone.0096967.

Yong VW, Chabot S, Stuve O, Williams G. Interferon beta in the treatment of multiple sclerosis: mechanisms of action. Neurology. 1998;51(3):682-689.

DOI: 10.1212/wnl.51.3.682.

Derynck R, Remaut E, Saman E, Stanssens P, De Clercq E, Content J, et al. Expression of human fibroblast interferon gene in Escherichia coli. Nature. 1980;287(5779):193-197. DOI: 10.1038/287193a0.

Rudick RA, Goelz SE. Beta-interferon for multiple sclerosis. Exp Cell Res. 2011;317(9):1301-1311. DOI: 10.1016/j.yexcr.2011.03.002.

Sadeghian-Rizi T, Ebrahimi A, Moazzen F, Yousefian H, Jahanian-Najafabadi A. Improvement of solubility and yield of recombinant protein expression in E. coli using a two-step system. Res Pharm Sci. 2019;14(5):400-407.

DOI: 10.4103/1735-5362.268200.

Matasci M, Hacker DL, Baldi L, Wurm FM. Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects. Drug Discov Today Technol. 2008;5(2-3):e37-42.

DOI: 10.1016/j.ddtec.2008.12.003.

Devasahayam M. Factors affecting the expression of recombinant glycoproteins. Indian J Med Res. 2007;126(1):22-27.

Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv. 2009;27(3):297-306.

DOI: 10.1016/j.biotechadv.2009.01.008.

Chung BK, Yusufi FN, Mariati, Yang Y, Lee DY. Enhanced expression of codon optimized interferon gamma in CHO cells. J Biotechnol. 2013;167(3):326-333.

DOI: 10.1016/j.jbiotec.2013.07.011.

Kim JY, Kim YG, Lee GM. CHO cells in biotechnology for production of recombinant proteins: current state and further potential. Appl Microbiol Biotechnol. 2012;93(3):917-930.

DOI: 10.1007/s00253-011-3758-5.

Jayapal KP, Wlaschin KF, Hu WS, Yap MGS. Recombinant protein therapeutics from CHO cells-20 years and counting. Chem Eng Prog. 2007;103(10):40-47.

Bandaranayake AD, Almo SC. Recent advances in mammalian protein production. FEBS Lett. 2014;588(2):253-260.

DOI: 10.1016/j.febslet.2013.11.035.

Kim CH, Oh Y, Lee TH. Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells. Gene. 1997;199(1-2):293-301. DOI: 10.1016/s0378-1119(97)00384-3.

Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004;22(11):1393-1398.

DOI: 10.1038/nbt1026.

Sharp PM, Li WH. The codon adaptation index-a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987;15(3):1281-1295.

DOI: 10.1093/nar/15.3.1281.

Kotsopoulou E, Bosteels H, Chim YT, Pegman P, Stephen G, Thornhill SI, et al. Optimised mammalian expression through the coupling of codon adaptation with gene amplification: maximum yields with minimum effort. J Biotechnol. 2010;146(4):186-193. DOI: 10.1016/j.jbiotec.2010.02.004.

Carton JM, Sauerwald T, Hawley-Nelson P, Morse B, Peffer N, Beck H, et al. Codon engineering for improved antibody expression in mammalian cells. Protein Expr Purif. 2007;55(2):279-286.

DOI: 10.1016/j.pep.2007.05.017.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685.

DOI: 10.1038/227680a0.

Poulain A, Mullick A, Massie B, Durocher Y. Reducing recombinant protein expression during CHO pool selection enhances frequency of high-producing cells. J Biotechnol. 2019;296:32-41.

DOI: 10.1016/j.jbiotec.2019.03.009.

Samoudi M, Minuchehr Z, Harcum SW, Tabandeh F, Omid Yeganeh N, Khodabandeh M. Rational design of glycoengineered interferon-β analogs with improved aggregation state: experimental validation. Protein Eng Des Sel. 2017;30(1):23-30.

DOI: 10.1093/protein/gzw058.

Han YK, Koo TY, Lee GM. Enhanced interferon‐beta production by CHO cells through elevated osmolality and reduced culture temperature. Biotechnol Prog. 2009;25(5):1440-1447.

DOI: 10.1002/btpr.234.

Ghavim M, Abnous K, Arasteh F, Taghavi S, Nabavinia MS, Alibolandi M, et al. High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region. Res Pharm Sci. 2017;12(2):168-175.

DOI: 10.4103/1735-5362.202462.

Kudla G, Lipinski L, Caffin F, Helwak A, Zylicz M. High guanine and cytosine content increases mRNA levels in mammalian cells. PLoS Biol. 2006;4(6):e180, 0933-0942.

DOI: 10.1371/journal.pbio.0040180.

Newman ZR, Young JM, Ingolia NT, Barton GM. Differences in codon bias and GC content contribute to the balanced expression of TLR7 and TLR9. Proc Natl Acad Sci U S A. 2016;113(10):E1362-E1371. DOI: 10.1073/pnas.1518976113.

Shimshek DR, Kim J, Hübner MR, Spergel DJ, Buchholz F, Casanova E, et al. Codon‐improved Cre recombinase (iCre) expression in the mouse. Genesis. 2002;32(1):19-26.

DOI: 10.1002/gene.10023.

Graf M, Bojak A, Deml L, Bieler K, Wolf H, Wagner R. Concerted action of multiple cis-acting sequences is required for Rev dependence of late human immunodeficiency virus type 1 gene expression. J Virol. 2000;74(22):10822-10826.

DOI: 10.1128/jvi.74.22.10822-10826.2000.


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