The Production of Recombinant Proteins from Mammalian Cells Using RNA Element

  • Intisar Fouad Ali Mursi
  • Seiji Masuda


Producing recombinant proteins in a large scale for pharmaceutical use is a challenging process as these proteins must be posttranscriptionally modified. Mammalian cells have proven to be good candidates for this process to take place efficiently. In order to optimize gene expression of the required proteins in mammalian cells, good vectors must be used such as the viral vectors. Vectors must be chosen cautiously according to the type of the mammalian cell line being utilized. Importantly, strong promoters must be selected to ensure large amounts of the gene(s) of interest.

The export of the messenger ribonucleic acid (mRNA) is a complex process in which many proteins are involved. A strategy to enhance recombinant protein production is to use the mRNA export pathway efficiently. In the mRNA export pathway, key proteins include the NXF1-NXT1 heterodimer. Here we introduce the use of constitutive transport element in the expression system. Constitutive transport element directly recruits mRNA export proteins NXF1-NXT1, and these events facilitate the mRNA export containing constitutive transport element. The simultaneous overexpression of mRNA export factors in addition to the use of RNA element recruiting mRNA export proteins is a potential strategy to obtain satisfactory amounts of the required proteins.


Recombinant protein production RNA export RNA element RNA-binding protein 



We would like to express our thanks to Lwando Moshani from Kyoto Seika University, Japan, for creating some of the drawings for this book chapter. This work was partially supported by JSPS KAKENHI Grant Number 17K19232 to SM.


  1. Aihara Y, Fujiwara N, Yamazaki T, Kambe T, Nagao M, Hirose Y, Masuda S (2011) Enhancing recombinant protein production in human cell lines with a constitutive transport element and mRNA export proteins. J Biotechnol 153:86–91CrossRefGoogle Scholar
  2. Almo SC, Love JD (2014) Better and faster: improvements and optimization for mammalian recombinant protein production. Curr Opin Struct Biol 26:39–43CrossRefGoogle Scholar
  3. Booth DS, Cheng Y, Frankel AD (2014) The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA. elife 3:e04121PubMedPubMedCentralGoogle Scholar
  4. Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, Hammarskjold ML (1994) A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication rev-independent. Proc Natl Acad Sci U S A 91:1256–1260CrossRefGoogle Scholar
  5. Caporilli S, Yu Y, Jiang J, White-Cooper H (2013) The RNA export factor, Nxt1, is required for tissue specific transcriptional regulation. PLoS Genet 9:e1003526CrossRefGoogle Scholar
  6. Coura S, Nardi NB (2008) A role for adeno-associated viral vectors in gene therapy. Genet Mol Biol 31:1–11CrossRefGoogle Scholar
  7. Das AT, Zhou X, Metz SW, Vink MA, Berkhout B (2016) Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells. Biotechnol J 11:71–79CrossRefGoogle Scholar
  8. Daya S, Berns KI (2008) Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 21:583–593CrossRefGoogle Scholar
  9. Delaleau M, Borden KLB (2015) Multiple export mechanisms for mRNAs. Cell 4:452–473CrossRefGoogle Scholar
  10. Deyle DR, Russell DW (2009) Adeno-associated virus vector integration. Curr Opin Mol Ther 11:442–447PubMedPubMedCentralGoogle Scholar
  11. Fassler M, Weissberg I, Levy N, Diaz-Griffero F, Monsonego A, Friedman A (2013) Preferential lentiviral targeting of astrocytes in the central nervous system. PLoS One 8:e76092CrossRefGoogle Scholar
  12. Fung HYJ, Fu S, Chook YM (2017) Nuclear export receptor CRM1 recognizes diverse conformations in nuclear export signals. elife 6:e23961CrossRefGoogle Scholar
  13. Gruenert AK, Czugala M, Mueller C, Schmeer M, Schleef M (2016) Self-complementary adeno-associated virus vectors improve transduction efficiency of corneal endothelial cells. PLoS One 11:e0152589CrossRefGoogle Scholar
  14. Hacker DL, Balasubramanian S (2016) Recombinant protein production from stable mammalian cell lines and pools. Curr Opin Struct Biol 38:129–136CrossRefGoogle Scholar
  15. Hendrickx R, Stichling N, Koelen J (2014) Innate immunity to adenovirus. Hum Gene Ther 25:265–284CrossRefGoogle Scholar
  16. Higby KJ, Bischak MM, Campbell CA, Anderson RG, Broskin SA, Foltz LE, Koper JA, Nickle AC, Resendes KK (2017) 5-Flurouracil disrupts nuclear export and nuclear pore permeability in a calcium dependent manner. Apoptosis 22:393–405CrossRefGoogle Scholar
  17. Hu Y (2005) Baculovirus as a highly efficient expression vector in insect and mammalian cells. Acta Pharmacol Sin 26:405–416CrossRefGoogle Scholar
  18. Hu WS, Aunins JG (1997) Large-scale mammalian cell culture. Curr Opin Biotechnol 8:148–153CrossRefGoogle Scholar
  19. Hutten S, Kehlenbach RH (2007) CRM1-mediated nuclear export: to the pore and beyond. Trends Cell Biol 17:193–201CrossRefGoogle Scholar
  20. Jackson DA, Symonst RH, Berg P (1972) Biochemical method for inserting new genetic information into DNA of Simian Virus 40: circular SV40 DNA molecules containing lambda phage genes and the galactose operon of Escherichia coli. Proc Natl Acad Sci U S A 69:2904–2909CrossRefGoogle Scholar
  21. Jakobsson J, Lundberg C (2006) Lentiviral vectors for use in the central nervous system. Mol Ther 13:484–493CrossRefGoogle Scholar
  22. Jeong S (2017) SR proteins : binders, regulators, and connectors of RNA. Mol Cells 40:1–9CrossRefGoogle Scholar
  23. Katahira J (2012) mRNA export and the TREX complex. Biochim Biophys Acta 819:507–513CrossRefGoogle Scholar
  24. Kędzierska H, Piekiełko-Witkowska A (2017) Splicing factors of SR and hnRNP families as regulators of apoptosis in cancer. Cancer Lett 396:53–65CrossRefGoogle Scholar
  25. Khan KH (2013) Gene expression in mammalian cells and its applications. Adv Pharm Bull 3:257–263PubMedPubMedCentralGoogle Scholar
  26. Kim H, Yoo SJ, Kang HA (2015) Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Res 15:1–16CrossRefGoogle Scholar
  27. Köhler A, Hurt E (2007) Exporting RNA from the nucleus to the cytoplasm. Nat Rev Mol Cell Biol 8:761–773CrossRefGoogle Scholar
  28. Kornblihtt AR, Schor IE, Alló M, Dujardin G, Petrillo E, Muñoz MJ (2013) Alternative splicing : a pivotal step between eukaryotic transcription and translation. Nat Rev Mol Cell Biol 14:153–166CrossRefGoogle Scholar
  29. Koyama M, Matsuura Y (2012) Mechanistic insights from the recent structures of the CRM1 nuclear export complex and its disassembly intermediate. Biophysics 8:145–150CrossRefGoogle Scholar
  30. Kurian KM, Watson CJ, Wyllie AH, Currie SA (2000) Retroviral vectors. Mol Pathol 53:173–176CrossRefGoogle Scholar
  31. Labow MA, Baim SB, Shenk T, Levine AJ (1990) Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells. Mol Cell Biol 10:3343–3356CrossRefGoogle Scholar
  32. Lee Y, Rio DC (2015) Mechanisms and regulation of alternative pre-mRNA splicing. Annu Rev Biochem 84:291–323CrossRefGoogle Scholar
  33. Li M, Husic N, Lin Y, Christensen H, Malik I, McIver S, LaPash Daniels CM, Harris DA, Kotzbauer PT, Goldberg MP, Snider BJ (2011) Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells. J Neurosci Methods 189:56–64CrossRefGoogle Scholar
  34. Li M, Husic N, Lin Y, Snider BJ (2012) Production of lentiviral vectors for transducing cells from the central nervous system. J Vis Exp 24:E4031Google Scholar
  35. Li Z, Michael IP, Zhou D, Nagy A, Rini JM (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc Natl Acad Sci U S A 110:5004–5009CrossRefGoogle Scholar
  36. Mamon LA, Ginanova VR, Kliver SF, Yakimova AO, Atsapkina AA, Golubkova EV (2017) RNA-binding proteins of the NXF (nuclear export factor) family and their connection with the cytoskeleton. Cytoskeleton (Hoboken) 74:161–169CrossRefGoogle Scholar
  37. Masuda S, Das R, Cheng H, Hurt E, Dorman N, Reed R (2005) Recruitment of the human TREX complex to mRNA during splicing. Genes Dev 19:1512–1517CrossRefGoogle Scholar
  38. McCarty DM, Monahan PE, Samulski RJ (2001) Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther 8:1248–1254CrossRefGoogle Scholar
  39. McCarty DM, Fu H, Monahan PE, Toulson CE, Naik P, Samulski RJ (2003) Adeno-associated virus terminal repeat (TR) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther 10:2112–2118CrossRefGoogle Scholar
  40. Mikami S, Masutani M, Sonenberg N, Yokoyama S, Imataka H (2006) An efficient mammalian cell-free translation system supplemented with translation factors. Protein Expr Purif 46:348–357CrossRefGoogle Scholar
  41. Müller-Mcnicoll M, Botti V, Domingues AMDJ, Brandl H, Schwich OD, Steiner MC, Curk T, Poser I, Zarnack K, Neugebauer KM (2016) SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export. Genes Dev 30:553–566CrossRefGoogle Scholar
  42. Nielsen J (2013) Production of biopharmaceutical proteins by yeast: advances through metabolic engineering. Bioengineered 4:207–211CrossRefGoogle Scholar
  43. Okamura M, Inose H, Masuda S (2015) RNA export through the NPC in eukaryotes. Genes 6:124–149CrossRefGoogle Scholar
  44. Onion D, Crompton LJ, Milligan DW, Moss PA, Lee SP, Mautner V (2007) The CD4+ T-cell response to adenovirus is focused against conserved residues within the hexon protein. J General Virol 88:2417–2425CrossRefGoogle Scholar
  45. Parr-Brownlie LC, Bosch-Bouju C, Schoderboeck L, Sizemore RJ, Abraham WC, Hughes SM (2015) Lentiviral vectors as tools to understand central nervous system biology in mammalian model organisms. Front Mol Neurosci 8:14CrossRefGoogle Scholar
  46. Pasquinelli AE, Ernst RK, Lund E, Grimm C, Zapp ML, Rekosh D, Hammarskjöld M, Dahlberg JE (1997) The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway. EMBO J 16:7500–7510CrossRefGoogle Scholar
  47. Qin JY, Zhang L, Clift KL, Hulur I, Xiang AP, Ren BZ, Lahn BT (2010) Systematic comparison of constitutive promoters and the doxycycline-inducible promoter. PLoS One 5:e10611CrossRefGoogle Scholar
  48. Quitschkes WW, Lin Z, Deponti-Zilli L, Paterson BM, Tris M (1989) The beta actin promoter. J Biol Chem 264:9539–9546Google Scholar
  49. Raj B, Blencowe BJ (2015) Alternative splicing in the mammalian nervous system : recent insights into mechanisms and functional roles. Neuron 87:14–27CrossRefGoogle Scholar
  50. Reed R (2003) Coupling transcription, splicing and mRNA export. Curr Opin Cell Biol 15:326–331CrossRefGoogle Scholar
  51. Reed R, Cheng H (2005) TREX, SR proteins and export of mRNA. Curr Opin Cell Biol 17:269–273CrossRefGoogle Scholar
  52. Rosenblum G, Cooperman BS (2014) Engine out of the chassis: cell-free protein synthesis and its uses. FEBS Lett 588:261–268CrossRefGoogle Scholar
  53. Roy B, Haupt LM, Griffiths LR (2013) Alternative splicing (AS) of genes as an approach for generating protein complexity. Curr Genomics 14:182–194CrossRefGoogle Scholar
  54. Sandri-goldin RM (2004) Viral regulation of mRNA export. J Virol 78:4389–4396CrossRefGoogle Scholar
  55. Schagen FH, Rademaker HJ, Fallaux FJ, Hoeben RC (2000) Insertion vectors for gene therapy. Gene Ther 7:271–272CrossRefGoogle Scholar
  56. Schorpp M, Jäger R, Schellander K, Schenkel J, Wagner EF, Weiher H, Angel P (1996) The human ubiquitin C promoter directs high ubiquitous expression of transgenes in mice. Nucleic Acids Res 24:1787–1788CrossRefGoogle Scholar
  57. Shao WY, Yang YL, Yan H, Huang Q, Liu KJ, Zhang S (2017) Phenethyl isothiocyanate suppresses the metastasis of ovarian cancer associated with the inhibition of CRM1-mediated nuclear export and mTOR-STAT3 pathway. Cancer Biol Ther 18:26–35CrossRefGoogle Scholar
  58. Strässer K, Masuda S, Mason P, Pfannstiel J, Oppizzi M, Rodriguez-Navarro S, Rondón AG, Aguilera A, Struhl K, Reed R, Hurt E (2002) TREX is a conserved complex coupling transcription with messenger RNA export. Nature 417:304–308CrossRefGoogle Scholar
  59. Tabernero C, Zolotukhin AS, Valentin A, Pavlakis GN, Felber BK (1996) The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function. J Virol 70:5998–6011PubMedPubMedCentralGoogle Scholar
  60. Turner JG, Dawson J, Sullivan DM (2012) Nuclear export of proteins and drug resistance in cancer. Biochem Pharmacol 83:1021–1032CrossRefGoogle Scholar
  61. Turner JG, Dawson J, Cubitt CL, Baz R, Sullivan DM (2014) Inhibition of CRM1-dependent nuclear export sensitizes malignant cells to cytotoxic and targeted agents. Semin Cancer Biol 27:62–73CrossRefGoogle Scholar
  62. Varela-Echavarría A, Prorock CM, Ron Y, Dougherty JP (1993) High rate of genetic rearrangement during replication of a Moloney murine leukemia virus-based vector. J Virol 67:6357–6364PubMedPubMedCentralGoogle Scholar
  63. Wickramasinghe VO, Laskey RA (2015) Control of mammalian gene expression by selective mRNA export. Nat Rev Mol Cell Biol 16:431–442CrossRefGoogle Scholar
  64. Wiegand HL, Coburn GA, Zeng Y, Kang Y, Bogerd HP, Cullen BR (2002) Formation of Tap/NXT1 heterodimers activates Tap-dependent nuclear mRNA export by enhancing recruitment to nuclear pore complexes. Mol Cell Biol 22:245–256CrossRefGoogle Scholar
  65. Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 22:1393–1398CrossRefGoogle Scholar
  66. Xu L, Daly T, Gao C, Flotte TR, Song S, Byrne BJ, Sands MS, Parker Ponder K (2001) CMV-beta-actin promoter directs higher expression from an adeno-associated viral vector in the liver than the cytomegalovirus or elongation factor 1 alpha promoter and results in therapeutic levels of human factor X in mice. Human Gene Ther 12:563–573Google Scholar
  67. Zhou S, Mody D, DeRavin SS, Hauer J, Lu T, Ma Z, Abina SH, Gray JT, Greene MR, Cavazzana-Calvo M, Malech HL, Sorrentino BP (2010) A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells. Blood 116:900–908CrossRefGoogle Scholar
  68. Zolotukhin S, Michalowski D, Smulevitch S, Felber BK (2001) Retroviral constitutive transport element evolved from cellular TAP(NXF1)-binding sequences. J Virol 75:5567–5575CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Division of Integrated Life Sciences, Graduate School of BiostudiesKyoto UniversityKyotoJapan

Personalised recommendations