Conceptual Models

  • Saeid KadkhodaeiEmail author
  • Farahnaz Sadat Golestan Hashemi
  • Morvarid Akhavan Rezaei
  • Sahar Abbasiliasi
  • Joo Shun Tan
  • Hamid Rajabi Memari
  • Faruku Bande
  • Ali Baradaran
  • Mahdi Moradpour
  • Arbakariya B. Ariff
Part of the SpringerBriefs in Systems Biology book series (BRIEFSBIOSYS)


In reviewing the literature, sometimes no or very little data are found on the specific expression elements for genetic engineering studies. This study set out with the aim of systematic discovery of the key upstream factors for genetic manipulation of microalgae as a case study. The review indicates how the MARs flanking the expression cassette along with the optimized expression elements could potentially improve the transformation efficiency and stability. The outlines can be efficiently deployed as a practical model for systematic discovery of the key expression elements and optimization of the cis/transgenes in other micro/organisms in which there is limited information about these factors.


Cis/transgene optimization Conceptual model Systematic discovery 


  1. 1.
    Agarwal S, Singh R, Sanyal I, Amla DV (2008) Expression of modified gene encoding functional human alpha-1-antitrypsin protein in transgenic tomato plants. Transgenic Res 17:881–896. Scholar
  2. 2.
    Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39. Scholar
  3. 3.
    Bellucci M, Alpini A, Paolocci F, Cong L, Arcioni S (2000) Accumulation of maize γ-zein and γ-zein: KDEL to high levels in tobacco leaves and differential increase of BiP synthesis in transformants. TAG Theor Appl Genet 101:796–804. Scholar
  4. 4.
    Cavener DR (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res 15:1353–1361. Scholar
  5. 5.
    Chang M, Liu R, Jin Q, Liu Y, Wang X (2014) Scaffold/Matrix attachment regions from CHO cell chromosome enhanced the stable transfection efficiency and the expression of transgene in CHO cells. Biotechnol Appl Biochem, 1–19Google Scholar
  6. 6.
    Chou K-C, Shen H-B (2007) Signal-CF: a subsite-coupled and window-fusing approach for predicting signal peptides. Biochem Biophys Res Commun 357:633–640. Scholar
  7. 7.
    Coragliotti AT, Beligni MV, Franklin SE, Mayfield SP (2011) Molecular factors affecting the accumulation of recombinant proteins in the Chlamydomonas reinhardtii chloroplast. Mol Biotechnol 48:60–75. Scholar
  8. 8.
    Díaz-Santos E, De La Vega M, Vila M, Vigara J, León R (2013) Efficiency of different heterologous promoters in the unicelullar microalga Chlamydomonas reinhardtii. Biotechnol Prog. Scholar
  9. 9.
    Eichler-Stahlberg A, Weisheit W, Ruecker O, Heitzer M (2009) Strategies to facilitate transgene expression in Chlamydomonas reinhardtii. Planta 229:873–883. Scholar
  10. 10.
    Emanuelsson O, Brunak S, von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–971. Scholar
  11. 11.
    Fukuda H, Arai M, Kuwajima K (2000) Folding of green fluorescent protein and the cycle3 mutant †. Biochemistry 39:12025–12032. Scholar
  12. 12.
    Fukuda Y, Nishikawa S (2003) Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease. Plant Mol Biol 51:665–675. Scholar
  13. 13.
    Gallie DR, Sleat DE, Watts JW, Turner PC, Wilson TMA (1987) A comparison of eukaryotic viral 5′-leader sequences as enhancers of mRNA expression in vivo. Nucleic Acids Res 15:8693–8711. Scholar
  14. 14.
    Gaspar P, Moura G, Santos MAS, Oliveira JL (2013) mRNA secondary structure optimization using a correlated stem-loop prediction. Nucleic Acids Res 41:e73. Scholar
  15. 15.
    Van der Geest AHM, Welter ME, Woosley AT, Pareddy DR, Pavelko SE, Skokut M, Ainley WM (2004) A short synthetic MAR positively affects transgene expression in rice and Arabidopsis. Plant Biotechnol J 2:13–26. Scholar
  16. 16.
    Geng D, Wang Y, Wang P, Li W, Sun Y (2003) Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta). J Appl Phycol 15:451–456. Scholar
  17. 17.
    Girod P, Nguyen D, Calabrese D, Puttini S, Grandjean M, Martinet D, Regamey A, Saugy D, Beckmann JS, Bucher P, Mermod N (2007) Genome-wide prediction of matrix attachment regions that increase gene expression in mammalian cells. Nat Methods 4:747–753. Scholar
  18. 18.
    Gomi M, Sonoyama M, Mitaku S (2004) High performance system for signal peptide prediction: SOSUI signal. Chem-Bio Informatics J 4:142–147. Scholar
  19. 19.
    Gorman C, Arope S, Grandjean M, Girod P, Mermod N (2009) Use of MAR Elements to Increase the Production of Recombinant Proteins. In: Al-Rubeai M (ed) Cell line development, cell engineering 6. Springer, Netherlands, Dordrecht, pp 1–32Google Scholar
  20. 20.
    Grandjean M, Girod P-A, Calabrese D, Kostyrko K, Wicht M, Yerly F, Mazza C, Beckmann JS, Martinet D, Mermod N (2011) High-level transgene expression by homologous recombination-mediated gene transfer. Nucleic Acids Res 39:1–15. Scholar
  21. 21.
    Gruber AR, Lorenz R, Bernhart SH, Neuböck R, Hofacker IL (2008) The Vienna RNA websuite. Nucleic Acids Res 36:W70–W74. Scholar
  22. 22.
    Grünert S, Jackson RJ (1994) The immediate downstream codon strongly influences the efficiency of utilization of eukaryotic translation initiation codons. EMBO J 13:3618–3630PubMedPubMedCentralCrossRefGoogle Scholar
  23. 23.
    Jamal A, Ko KK, Kim H, Choo Y, Joung H, Ko KK (2009) Role of genetic factors and environmental conditions in recombinant protein production for molecular farming. Biotechnol Adv 27:914–923. Scholar
  24. 24.
    Joshi CP, Zhou H, Huang X, Chiang VL (1997) Context sequences of translation initiation codon in plants. Plant Mol Biol 35:993–1001. Scholar
  25. 25.
    Kadkhodaei S, Abbasiliasi S, Shun TJ, Fard Masoumi HR, Mohamed MS, Movahedi A, Rahim R, Ariff AB, Masoumi HRF, Mohamed MS, Movahedi A, Rahim R, Ariff AB (2015) Enhancement of protein production by microalgae Dunaliella salina under mixotrophic conditions using response surface methodology. RSC Adv 5:38141–38151. Scholar
  26. 26.
    Kozak M (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 196:947–950. Scholar
  27. 27.
    Kozak M (1990) Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes. Proc Natl Acad Sci 87:8301–8305. Scholar
  28. 28.
    Kozak M (1997) Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positions +5 and +6. EMBO J 16:2482–2492. Scholar
  29. 29.
    Ley D, Harraghy N, Le Fourn V, Bire S, Girod P-A, Regamey A, Rouleux-Bonnin F, Bigot Y, Mermod N (2013) MAR elements and transposons for improved transgene integration and expression. PLoS ONE 8:e62784. Scholar
  30. 30.
    Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Pabst M, Castilho A, Kunert R, Liang M, Arcalis E, Robinson DG, Depicker A, Steinkellner H (2011) Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Plant Physiol 155:2036–2048. Scholar
  31. 31.
    Lukaszewicz M, Feuermann M, Jérouville B, Stas A, Boutry M (2000) In vivo evaluation of the context sequence of the translation initiation codon in plants. Plant Sci 154:89–98. Scholar
  32. 32.
    Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–48PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Maertens B, Spriestersbach A, von Groll U, Roth U, Kubicek J, Gerrits M, Graf M, Liss M, Daubert D, Wagner R, Schäfer F (2010) Gene optimization mechanisms: a multi-gene study reveals a high success rate of full-length human proteins expressed in Escherichia coli. Protein Sci 19:1312–1326. Scholar
  34. 34.
    Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH (2004) Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101:7287–7292. Scholar
  35. 35.
    Matsukawa S, Moriyama Y, Hayata T, Sasaki H, Ito Y, Asashima M, Kuroda H (2012) KDEL tagging: a method for generating dominant-negative inhibitors of the secretion of TGF-beta superfamily proteins. Int J Dev Biol 56:351–356. Scholar
  36. 36.
    Menne KML, Hermjakob H, Apweiler R (2000) A comparison of signal sequence prediction methods using a test set of signal peptides. Bioinformatics 16:741–742. Scholar
  37. 37.
    Nakagawa S, Niimura Y, Gojobori T, Tanaka H, Miura K (2008) Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes. Nucleic Acids Res 36:861–871. Scholar
  38. 38.
    Nielsen H, Krogh A (1998) Prediction of signal peptides and signal anchors by a hidden Markov model. Intell Syst Mol Biol 6:122–130. doi: Scholar
  39. 39.
    Nowak W, Gawłowska M, Jarmołowski A, Augustyniak J (2001) Effect of nuclear matrix attachment regions on transgene expression in tobacco plants. Acta Biochim Pol 48:637–646PubMedGoogle Scholar
  40. 40.
    Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. Scholar
  41. 41.
    Petruccelli S, Otegui MS, Lareu F, Tran Dinh O, Fitchette A-C, Circosta A, Rumbo M, Bardor M, Carcamo R, Gomord V, Beachy RN (2006) A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J 4:511–527. Scholar
  42. 42.
    Pisarev AV (2006) Specific functional interactions of nucleotides at key −3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex. Genes Dev 20:624–636. Scholar
  43. 43.
    Potvin G, Zhang Z (2010) Strategies for high-level recombinant protein expression in transgenic microalgae: a review. Biotechnol Adv 28:910–918. Scholar
  44. 44.
    Puigbò P, Aragonès L, Garcia-Vallvé S (2010) RCDI/eRCDI: a web-server to estimate codon usage deoptimization. BMC Res Notes 3:87. Scholar
  45. 45.
    Rabani M, Kertesz M, Segal E (2011) Computational prediction of RNA structural motifs involved in post-transcriptional regulatory processes. Methods Mol Biol 714:467–479CrossRefPubMedGoogle Scholar
  46. 46.
    Ramanan RN, Wong BT, Memari HR, Azaman SNA, Tau CL, Beng TT, Mohd Lila MA, Abdullah MP, Abdul Rahim R, Ariff A (2010) Effect of promoter strength and signal sequence on the periplasmic expression of human interferon-2b in Escherichia coli. African J Biotechnol 9:285–292Google Scholar
  47. 47.
    Reynolds SM, Käll L, Riffle ME, Bilmes JA, Noble WS (2008) Transmembrane topology and signal peptide prediction using dynamic bayesian networks. PLoS Comput Biol 4:e1000213. Scholar
  48. 48.
    Satchidanandam V, Shivashankar Y (1997) Availability of a second upstream AUG can completely overcome inhibition of protein synthesis initiation engendered by mRNA secondary structure encompassing the start codon. Gene 196:231–237. Scholar
  49. 49.
    Sawant SV (2001) Sequence architecture downstream of the initiator codon enhances gene expression and protein stability in plants. Plant Physiol 126:1630–1636. Scholar
  50. 50.
    Schouten A, Roosien J, van Engelen FA, (Ineke) de Jong GAM, (Tanja) Borst-Vrenssen AWM, Zilverentant JF, Bosch D, Stiekema WJ, Gommers FJ, Schots A, Bakker J (1996) The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol Biol 30:781–793.
  51. 51.
    Schroda M, Beck CF, Vallon O (2002) Sequence elements within an HSP70 promoter counteract transcriptional transgene silencing in Chlamydomonas. Plant J 31:445–455. Scholar
  52. 52.
    Schroda M, Blocker D, Beck CF (2000) The HSP70A promoter as a tool for the improved expression of transgenes in Chlamydomonas. Plant J 21:121–131. Scholar
  53. 53.
    Shen H-B, Chou K-C (2007) Signal-3L: a 3-layer approach for predicting signal peptides. Biochem Biophys Res Commun 363:297–303. Scholar
  54. 54.
    Singh G (1997) Mathematical model to predict regions of chromatin attachment to the nuclear matrix. Nucleic Acids Res 25:1419–1425. Scholar
  55. 55.
    Sleat DE, Gallic DR, Jefferson RA, Bevan MW, Turner PC, Wilson TMA (1987) Characterisation of the 5′-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro. Gene 60:217–225. Scholar
  56. 56.
    de Smit MH, van Duin J (1990) Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci 87:7668–7672. Scholar
  57. 57.
    Sun Y, Yang Z, Gao X, Li Q, Zhang Q, Xu Z (2005) Expression of foreign genes in Dunaliella by electroporation. Mol Biotechnol 30:185–192. Scholar
  58. 58.
    Theis C, Janssen S, Giegerich R (2010) Prediction of RNA secondary structure including kissing hairpin motifs. In: Algorithms in bioinformatics. pp 52–64Google Scholar
  59. 59.
    Vain P, Worland B, Kohli A, Snape JW, Christou P, Allen GC, Thompson WF (1999) Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J 18:233–242. Scholar
  60. 60.
    Viklund H, Bernsel A, Skwark M, Elofsson A (2008) SPOCTOPUS: a combined predictor of signal peptides and membrane protein topology. Bioinformatics 24:2928–2929. Scholar
  61. 61.
    Vimberg V, Tats A, Remm M, Tenson T (2007) Translation initiation region sequence preferences in Escherichia coli. BMC Mol Biol 8:100. Scholar
  62. 62.
    Walker T (2004) The development of microalgae as a bioreactor system for the production of recombinant proteins. Queensland University of TechnologyGoogle Scholar
  63. 63.
    Wang A, Ma S (2012) Molecular farming in plants: Recent advances and future prospects. Springer, Netherlands, DordrechtCrossRefGoogle Scholar
  64. 64.
    Wang T, Hou G, Wang Y, Xue L (2010) Characterization and heterologous expression of a new matrix attachment region binding protein from the unicellular green alga Dunaliella salina. J Biochem 148:651–658. Scholar
  65. 65.
    Xu C, Lin H, Likbi P, Jean M (2011) Polymer encapsulated aluminum particulates. WO Pat 2,011,068,596 1Google Scholar
  66. 66.
    Xue H, Yang Y-T, Wu C-A, Yang G-D, Zhang M-M, Zheng C-C (2005) TM2, a novel strong matrix attachment region isolated from tobacco, increases transgene expression in transgenic rice calli and plants. Theor Appl Genet 110:620–627. Scholar
  67. 67.
    Zalucki YM, Power PM, Jennings MP (2007) Selection for efficient translation initiation biases codon usage at second amino acid position in secretory proteins. Nucleic Acids Res 35:5748–5754. Scholar
  68. 68.
    Zhang W, Xiao W, Wei H, Zhang J, Tian Z (2006) mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli. Biochem Biophys Res Commun 349:69–78. Scholar
  69. 69.
    Zimmermann R, Eyrisch S, Ahmad M, Helms V (2011) Protein translocation across the ER membrane. Biochim Biophys Acta 1808:912–924. Scholar
  70. 70.
    (2011) Manual for CLC Main Workbench 6.0 Google Scholar

Copyright information

© The Author(s) 2018

Authors and Affiliations

  • Saeid Kadkhodaei
    • 1
    Email author
  • Farahnaz Sadat Golestan Hashemi
    • 2
  • Morvarid Akhavan Rezaei
    • 3
  • Sahar Abbasiliasi
    • 4
  • Joo Shun Tan
    • 5
  • Hamid Rajabi Memari
    • 6
  • Faruku Bande
    • 7
  • Ali Baradaran
    • 8
    • 9
  • Mahdi Moradpour
    • 10
  • Arbakariya B. Ariff
    • 11
  1. 1.Research Institute for Biotechnology and BioengineeringIsfahan University of TechnologyIsfahanIran
  2. 2.Plant Genetics, AgroBioChem Department, Gembloux Agro-Bio TechUniversity of LiègeLiègeBelgium
  3. 3.Tropical Infectious Diseases Research and Education Centre (TIDREC), Faculty of MedicineUniversity of MalayaKuala LumpurMalaysia
  4. 4.Halal Products Research InstituteUniversiti Putra MalaysiaSeri KembanganMalaysia
  5. 5.Bioprocess Technology, School of Industrial TechnologyUniversiti Sains MalaysiaGeorge Town, PenangMalaysia
  6. 6.SynHiTechThornhillCanada
  7. 7.Department of Veterinary Services, Ministry of Animal Health and Fisheries DevelopmentUsman Faruk Secretariat, SokotoSokotoNigeria
  8. 8.Mater ResearchTranslational Research InstituteWoolloongabbaAustralia
  9. 9.Faculty of Medicine, Translational Research Institute, Diamantina InstituteUniversity of QueenslandBrisbaneAustralia
  10. 10.Institute of plantation studiesUniversiti Putra MalaysiaSeri KembanganMalaysia
  11. 11.Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaSeri KembanganMalaysia

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