Skip to main content

Advertisement

SpringerLink
Log in

Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells

  • Review
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Promoters are essential on plasmid vectors to initiate transcription of the transgenes when generating therapeutic recombinant proteins expressing mammalian cell lines. High and sustained levels of gene expression are desired during therapeutic protein production while gene expression is useful for cell engineering. As many finely controlled promoters exhibit cell and product specificity, new promoters need to be identified, optimized and carefully evaluated before use. Suitable promoters can be identified using techniques ranging from simple molecular biology methods to modern high-throughput omics screenings. Promoter engineering is often required after identification to either obtain high and sustained expression or to provide a wider range of gene expression. This review discusses some of the available methods to identify and engineer promoters for therapeutic recombinant protein expression in mammalian cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1

Similar content being viewed by others

References

  • Abeel T, Saeys Y, Bonnet E, Rouzé P, Van De Peer Y (2008) Generic eukaryotic core promoter prediction using structural features of DNA. Genome Res 18:310–323

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Addison CL, Hitt M, Kunsken D, Graham FL (1997) Comparison of the human versus murine cytomegalovirus immediate early gene promoters for transgene expression by adenoviral vectors. J Gen Virol 78:1653–1660

    CAS  PubMed  Google Scholar 

  • Aggarwal S (2012) What’s fueling the biotech engine—2011 to 2012. Nat Biotechnol 30:1191–1197

    Article  CAS  PubMed  Google Scholar 

  • Barnes LM, Bentley CM, Dickson AJ (2003) Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng 81:631–639

    Article  CAS  PubMed  Google Scholar 

  • Birch JR, Racher AJ (2006) Antibody production. Adv Drug Deliv Rev 58:671–685

    Article  CAS  PubMed  Google Scholar 

  • Blazeck J, Alper HS (2013) Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J 8:46–58

    Article  CAS  PubMed  Google Scholar 

  • Blow MJ, McCulley DJ, Li Z, Zhang T, Akiyama JA et al (2010) ChIP-seq identification of weakly conserved heart enhancers. Nat Genet 42:806–810

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Boshart M, Weber F, Jahn G (1985) A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41:521–530

    Article  CAS  PubMed  Google Scholar 

  • Bulger M, Groudine M (2010) Enhancers: the abundance and function of regulatory sequences beyond promoters. Dev Biol 339:250–257

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chao SH, Harada JN, Hyndman F, Gao X, Nelson CG et al (2004) PDX1, a cellular homeoprotein, binds to and regulates the activity of human cytomegalovirus immediate early promoter. J Biol Chem 279:16111–16120

    Article  CAS  PubMed  Google Scholar 

  • Chen J, Haverty J, Deng L, Li GH, Qiu P et al (2013) Identification of a novel endogenous regulatory element in Chinese hamster ovary cells by promoter trap. J Biotechnol 167:255–261

    Article  CAS  PubMed  Google Scholar 

  • Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P et al (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196

    Article  CAS  PubMed  Google Scholar 

  • Clapier CR, Cairns BR (2009) The biology of chromatin remodeling complexes. Annu Rev Biochem 78:273–304

    Article  CAS  PubMed  Google Scholar 

  • Costa AR, Rodrigues ME, Henriques M, Azeredo J, Oliveira R (2010) Guidelines to cell engineering for monoclonal antibody production. Eur J Pharm Biopharm 74:127–138

    Article  Google Scholar 

  • Deer JR, Allison DS (2004) High-level expression of proteins in mammalian cells using transcription regulatory sequences from the Chinese hamster EF-1α gene. Biotechnol Prog 20:880–889

    Article  CAS  Google Scholar 

  • Dorsch-Hasler K, Keil GM, Weber F (1985) A long and complex enhancer activates transcription of the gene coding for the highly abundant immediate early mRNA in murine cytomegalovirus. Proc Natl Acad Sci USA 82:8325–8329

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Fan L, Kadura I, Krebs LE, Larson J, Bowden D et al (2013) Development of a highly-efficient CHO cell line generation system with engineered SV40E promoter. J Biotechnol 168:652–658

    Article  CAS  PubMed  Google Scholar 

  • Furey TS (2012) ChIP-seq and beyond: new and improved methodologies to detect and characterize protein–DNA interactions. Nat Rev Genet 13:840–852

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Gan Y, Guan J, Zhou S (2012) A comparison study on feature selection of DNA structural properties for promoter prediction. BMC Bioinform 13:4

    Article  Google Scholar 

  • Giresi PG, Kim J, McDaniell RM, Iyer VR, Lieb JD (2007) FAIRE (formaldehyde-assisted isolation of regulatory elements) isolates active regulatory elements from human chromatin. Genome Res 17:877–885

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Grabherr MG, Pontiller J, Mauceli E, Ernst W, Baumann M et al (2011) Exploiting nucleotide composition to engineer promoters. PLoS ONE 6:e20136

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ho SCL, Tong YW, Yang Y (2013) Generation of monoclonal antibody-producing mammalian cell lines. Pharm Bioprocess 1:71–87

    Article  Google Scholar 

  • Juven-Gershon T, Kadonaga JT (2010) Regulation of gene expression via the core promoter and the basal transcriptional machinery. Dev Biol 339:225–229

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Kalwy S (2005) Towards stronger gene expression—a promoter’s tale. In: 19th European Society for Animal Cell Technology (ESACT) meeting, Harrogate, England

  • Kang M, Kim SY, Lee S, Lee YK, Lee J et al (2005) Human β-globin second intron highly enhances expression of foreign genes from murine cytomegalovirus immediate-early promoter. J Microbiol Biotechnol 15:544–550

    CAS  Google Scholar 

  • Kim DW, Uetsuki T, Kaziro Y, Yamaguchi N, Sugano S (1990) Use of the human elongation factor-1-alpha promoter as a versatile and efficient expression system. Gene 91:217–223

    Article  CAS  PubMed  Google Scholar 

  • Kim SY, Lee JH, Shin HS, Kang HJ, Kim YS (2002) The human elongation factor 1 alpha (EF-1α) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter. J Biotechnol 93:183–187

    Article  CAS  PubMed  Google Scholar 

  • Kim M, O’Callaghan PM, Droms KA, James DC (2011) A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies. Biotechnol Bioeng 108:2434–2446

    Article  CAS  Google Scholar 

  • Kling J (2012) Fresh from the biotech pipeline 2011. Nat Biotechnol 30:128–131

    Article  CAS  PubMed  Google Scholar 

  • Kuczewski M, Schirmer E, Lain B, Zarbis-Papastoitsis G (2011) A single-use purification process for the production of a monoclonal antibody produced in a PER.C6 human cell line. Biotechnol J 6:56–65

    Article  CAS  PubMed  Google Scholar 

  • Kwaks TH, Sewalt RG, van Blokland R, Siersma TJ, Kasiem M et al (2005) Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells. J Biotechnol 115:35–46

    Article  CAS  PubMed  Google Scholar 

  • Le Hir H, Nott A, Moore MJ (2003) How introns influence and enhance eukaryotic gene expression. Trends Biochem Sci 28:215–220

    Article  PubMed  Google Scholar 

  • Le H, Vishwanathan N, Kantardjieff A, Doo I, Srienc M et al (2013) Dynamic gene expression for metabolic engineering of mammalian cells in culture. Metab Eng 20:212–220

    Article  CAS  PubMed  Google Scholar 

  • Lee D, Karchin R, Beer MA (2011) Discriminative prediction of mammalian enhancers from DNA sequence. Genome Res 21:2167–2180

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • LeProust EM, Peck BJ, Spirin K, McCuen HB, Moore B et al (2010) Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res 38:2522–2540

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lin H, Li QZ (2011) Eukaryotic and prokaryotic promoter prediction using hybrid approach. Theor Biosci 130:91–100

    Article  Google Scholar 

  • Magnusson T, Haase R, Schleef M, Wagner E, Ogris M (2011) Sustained, high transgene expression in liver with plasmid vectors using optimized promoter–enhancer combinations. J Gen Med 13:382–391

    Article  CAS  Google Scholar 

  • Malik S, Roeder RG (2010) The metazoan mediator co-activator complex as an integrative hub for transcriptional regulation. Nat Rev Genet 11:761–772

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mariati Ng YK, Chao SH, Yap MG, Yang Y (2010) Evaluating regulatory elements of human cytomegalovirus major immediate early gene for enhancing transgene expression levels in CHO K1 and HEK293 cells. J Biotechnol 147:160–163

    Article  CAS  PubMed  Google Scholar 

  • Melnikov A, Murugan A, Zhang X, Tesileanu T, Wang L et al (2012) Systematic dissection and optimization of inducible enhancers in human cells using a massively parallel reporter assay. Nat Biotechnol 30:271–277

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nelson JA, Reynolds-Kohler C, Smith BA (1987) Negative and positive regulation by a short segment in the 5′-flanking region of the human cytomegalovirus major immediate-early gene. Mol Cell Biol 7:4125–4129

    CAS  PubMed Central  PubMed  Google Scholar 

  • Niwa H, Yamamura K, Miyazaki J (1991) Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–200

    Article  CAS  PubMed  Google Scholar 

  • Nott A, Meislin SH, Moore MJ (2003) A quantitative analysis of intron effects on mammalian gene expression. RNA 9:607–617

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ogawa R, Kagiya G, Kodaki T, Fukuda S, Yamamoto K (2007) Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42:628–633

    Article  CAS  PubMed  Google Scholar 

  • Ong CT, Corces VG (2011) Enhancer function: new insights into the regulation of tissue-specific gene expression. Nat Rev Genet 12:283–293

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Osterlehner A, Simmeth S, Göpfert U (2011) Promoter methylation and transgene copy numbers predict unstable protein production in recombinant chinese hamster ovary cell lines. Biotechnol Bioeng 108:2670–2681

    Article  CAS  PubMed  Google Scholar 

  • Patwardhan RP, Hiatt JB, Witten DM, Kim MJ, Smith RP et al (2012) Massively parallel functional dissection of mammalian enhancers in vivo. Nat Biotechnol 30:265–270

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pedersen AG, Baldi P, Chauvin Y, Brunak S (1999) The biology of eukaryotic promoter prediction—a review. Comput Chem 23:191–207

    Article  CAS  PubMed  Google Scholar 

  • Pham P, Kamen A, Durocher Y (2006) Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol Biotechnol 34:225–237

    Article  CAS  PubMed  Google Scholar 

  • Pontiller J, Gross S, Thaisuchat H, Hesse F, Ernst W (2008) Identification of CHO endogenous promoter elements based on a genomic library approach. Mol Biotechnol 39:135–139

    Article  CAS  PubMed  Google Scholar 

  • Pontiller J, Maccani A, Baumann M, Klancnik I, Ernst W (2010) Identification of CHO endogenous gene regulatory elements. Mol Biotechnol 45:235–240

    Article  CAS  PubMed  Google Scholar 

  • Prentice HL, Tonkin CJD, Caamano L, Sisk WP (2007) High level expression of proteins using sequences from the ferritin heavy chain gene locus. J Biotechnol 128:50–60

    Article  CAS  PubMed  Google Scholar 

  • Quilici LS, Silva-Pereira I, Andrade AC, Albuquerque FC, Brigido MM et al (2013) A minimal cytomegalovirus intron A variant can improve transgene expression in different mammalian cell lines. Biotechnol Lett 35:21–27

    Article  CAS  PubMed  Google Scholar 

  • Reik A, Zhou Y, Collingwood TN, Warfe L, Bartsevich V et al (2007) Enhanced protein production by engineered zinc finger proteins. Biotechnol Bioeng 97:1180–1189

    Article  CAS  PubMed  Google Scholar 

  • Schlabach MR, Hu JK, Li M, Elledge SJ (2010) Synthetic design of strong promoters. Proc Natl Acad Sci USA 107:2538–2543

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smale ST, Kadonaga JT (2003) The RNA polymerase II core promoter. Annu Rev Biochem 72:449–479

    Article  CAS  PubMed  Google Scholar 

  • Song L, Zhang Z, Grasfeder LL, Boyle AP, Giresi PG et al (2011) Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity. Genome Res 21:1757–1767

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sumitomo Y, Higashitsuji H, Liu Y, Fujita T, Sakurai T et al (2012) Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells. BMC Biotechnol 12(1):72

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Swindle CS, Kim HG, Klug CA (2004) Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells. J Biol Chem 279:34–41

    Article  CAS  PubMed  Google Scholar 

  • Thaisuchat H, Baumann M, Pontiller J, Hesse F, Ernst W (2011) Identification of a novel temperature sensitive promoter in CHO cells. BMC Biotechnol 11(1):51

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Thomsen DR, Stenberg RM, Goins WF, Stinski MF (1984) Promoter-regulatory region of the major immediate early gene of human cytomegalovirus. Proc Natl Acad Sci USA 81:659–663

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Uetsuki T, Naito A, Nagata S, Kaziro Y (1989) Isolation and characterization of the human chromosomal gene for polypeptide chain elongation factor-1 alpha. J Biol Chem 264:5791–5798

    CAS  PubMed  Google Scholar 

  • Weber F, de Villiers J, Schaffner W (1984) An SV40 “enhancer trap” incorporates exogenous enhancers or generates enhancers from its own sequences. Cell 36:983–992

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Xia W, Bringmann P, McClary J, Jones PP, Manzana W et al (2006) High levels of protein expression using different mammalian CMV promoters in several cell lines. Protein Expr Purif 45:115–124

    Article  CAS  PubMed  Google Scholar 

  • Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z et al (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol 29:735–741

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang Y, Mariati Chusainow J, Yap MG (2010) DNA methylation contributes to loss in productivity of monoclonal antibody-producing CHO cell lines. J Biotechnol 147:180–185

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgment

This work was supported by the Biomedical Research Council/Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore.

Author information

Authors and Affiliations

  1. Agency for Science, Technology and Research (A*STAR), Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, Singapore, 138668, Singapore

    Steven C. L. Ho & Yuansheng Yang

  2. Division of Bioengineering, Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, Singapore, 119620, Singapore

    Steven C. L. Ho

  3. School of Chemical and Biomedical Engineering, Nanyang Technological University, N1.2-B2-33, 62 Nanyang Avenue, Singapore, 637459, Singapore

    Yuansheng Yang

Authors
  1. Steven C. L. Ho
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuansheng Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuansheng Yang.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ho, S.C.L., Yang, Y. Identifying and engineering promoters for high level and sustainable therapeutic recombinant protein production in cultured mammalian cells. Biotechnol Lett 36, 1569–1579 (2014). https://doi.org/10.1007/s10529-014-1523-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-014-1523-4

Keywords

Search

Navigation