Recombinant Protein Expression Optimizing: A Review of S/MAR, STAR, and UCOE, as a Chromatin-Modifying Element

Abstract

The rapid growth of the global biopharmaceutical market in recent years indicates its importance in the biotechnology industry. The production of these medicinal products is one of the fastest-growing industries in the patient treatment field. Since 1980, processes have been developed to optimize the production of recombinant protein products upstream and downstream. In this regard, one promising approach is the engineering of expression vectors based on combinations of DNA regulatory elements found in euchromatin regions. This approach aims to achieve proper gene integration and facilitate its expression in the target cell, as epigenetic mechanisms can lead to instability of the desired gene in long-term cell cultures and gene silencing. To address this issue, genetically engineered vectors have been produced, which include components such as Matrix Attachment Regions (MARs), Scaffold Attachment Regions (SARs), Stabilizing Anti-Repressor Elements (STAREs), and Ubiquitous Chromatin Opening Elements (UCOE). These components can modify the chromatin environment to minimize gene silencing and enhance higher, more stable, and biologically active expression of recombinant molecules. In this review, we focus on different approaches and developments in the technology of expression vector engineering and their impact on increasing the production of recombinant proteins.

Keywords:

recombinant protein, biopharmaceuticals, chromatin-modifying elements, UCOE, S/MAR, STAR

References
[1] Morrison CJNB. Fresh from the biotech pipeline— 2018. Nat Biotechnol. 2019;37(2):118-23.

[2] Muralidhara BK, Wong M. Critical considerations in the formulation development of parenteral biologic drugs. Drug Discov Today. 2020;25(3):574-81.

[3] Aggarwal RS. What’s fueling the biotech engine- 2012 to 2013. Nat Biotechnol. 2014;32(1):32-9.

[4] Kaplon H, Reichert JM. Antibodies to watch in 2019. mAbs. 2019;11(2):219-38.

[5] Portolano N, Watson PJ, Fairall L, Millard CJ, Milano CP, Song Y, et al. Recombinant protein expression for structural biology in HEK 293F suspension cells: A novel and accessible approach. J Vis Exp. 2014(92):e51897.

[6] Li Q, Zhao Cp, Lin Y, Song C, Wang F, Wang TyJJoc, et al. Two human MAR s effectively increase transgene expression in transfected CHO cells. J Cell Mol Med. 2019;23(2):1613-6.

[7] 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):917-30.

[8] Dahodwala H, Lee KHJCoib. The fickle CHO: A review of the causes, implications, and potential alleviation of the CHO cell line instability problem. Curr Opin Biotechnol. 2019;60:128-37.

[9] Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004;22(11):1393-8.

[10] Barnes LM, Bentley CM, Dickson AJ. Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng. 2003;81(6):631-9.

[11] Hamaker NK, Lee KH. Site-specific Integration Ushers in a New Era of Precise CHO Cell Line Engineering. Curr Opin Chem Eng. 2018;22:152-60.

[12] Wright WD, Shah SS, Heyer WD. Homologous recombination and the repair of DNA double-strand breaks. J Biol Chem. 2018;293(27):10524-35.

[13] Kalkan AK, Palaz F, Sofija S, Elmousa N, Ledezma Y, Cachat E, et al. Improving recombinant protein production in CHO cells using the CRISPR-Cas system. Biotechnol Adv. 2023;64:108115.

[14] Wurm FM, Wurm MJ. Cloning of CHO cells, productivity and genetic stability—A discussion. Processes. 2017;5(2):20.

[15] Lee JS, Kildegaard HF, Lewis NE, Lee GM. Mitigating Clonal Variation in Recombinant Mammalian Cell Lines. Trends Biotechnol. 2019;37(9):931-42.

[16] Hamaker NK, Lee KH. Site-specific integration ushers in a new era of precise CHO cell line engineering. Current opinion in chemical engineering. Curr Opin Chem Eng. 2018;22:152-60.

[17] Li Q, Yan RF, Yang YX, Mi CL, Jia YL, Wang TY. Stabilizing and anti-repressor elements effectively increases transgene expression in transfected CHO Cells. Front Bioeng Biotechnol. 2022;10:840600.

[18] Romanova N, Noll T. Engineered and natural promoters and chromatin-modifying elements for recombinant protein expression in CHO Cells. Biotechnol J. 2018;13(3):e1700232.

[19] Guo X, Wang C, Wang TY. Chromatin-modifying elements for recombinant protein production in mammalian cell systems. Critical Reviews in Biotechnology. 2020;40(7):1035-43.

[20] Arope S, Harraghy N, Pjanic M, Mermod N. Molecular characterization of a human matrix attachment region epigenetic regulator. PloS One. 2013;8(11):e79262.

[21] Narwade N, Patel S, Alam A, Chattopadhyay S, Mittal S, Kulkarni A. Mapping of scaffold/matrix attachment regions in human genome: A data mining exercise. Nucleic Acids Res. 2019;47(14):7247-61.

[22] Kim J, Kollhoff A, Bergmann A, Stubbs L. Methylation-sensitive binding of transcription factor YY1 to an insulator sequence within the paternally expressed imprinted gene, Peg3. Hum Mol Genet. 2003;12(3):233-45.

[23] Chang M, Liu R, Jin Q, Liu Y, Wang X. Scaffold/matrix attachment regions from CHO cell chromosome enhanced the stable transfection efficiency and the expression of transgene in CHO cells. Biotechnol Appl Bioc. 2014;61(5):510-6.

[24] Kim HY, editor, Improved expression vector activity using insulators and scaffold / matrix-attachment regions for enhancing recombinant protein production. BioProcess International. 2006.

[25] Kwaks TH, Barnett P, Hemrika W, Siersma T, Sewalt RG, Satijn DP, et al. Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nat Biotechnol. 2003;21(5):553-8.

[26] Hoeksema F, van Blokland R, Siep M, Hamer K, Siersma T, den Blaauwen J, et al. The use of a stringent selection system allows the identification of DNA elements that augment gene expression. Mol Biotechnol. 2011;48(1):19-29.

[27] Williams S, Mustoe T, Mulcahy T, Griffiths M, Simpson D, Antoniou M, et al. CpG-island fragments from the HNRPA2B1/CBX3 genomic locus reduce silencing and enhance transgene expression from the hCMV promoter/enhancer in mammalian cells. BMC Biotechnol. 2005;5:17.

[28] Sizer RE, White RJ. Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology. Comput Struct Biotechnol J. 2023;21:275-83.

[29] Boscolo S, Mion F, Licciulli M, Macor P, De Maso L, Brce M, et al. Simple scale-up of recombinant antibody production using an UCOE containing vector. New Biotechnol. 2012;29(4):477-84.

[30] Skipper KA, Hollensen AK, Antoniou MN, Mikkelsen JGJBb. Sustained transgene expression from sleeping beauty DNA transposons containing a core fragment of the HNRPA2B1-CBX3 ubiquitous chromatin opening element (UCOE). BMC Biotechnol.2019;19:1- 11.

[31] Hoseinpoor R, Kazemi B, Rajabibazl M, Rahimpour AJJoB. Improving the expression of anti-IL-2R