Skip to main content

Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection


Scanning DNA sequences for polymorphisms and mutations often involve the mismatch specific cleavage by endonucleases at the mismatch sites and subsequent analysis of the digested product for mutation discovery. One of the limitations of using enzymatic mutation detection methods are the cost and availability of a mismatch specific endonuclease. We report the establishment of Nicotiana tabacum L. Cv. Bright Yellow 2 cells stably expressing the truncated ENDO1 (tENDO1) mismatch specific endonuclease. The 5′-Untranslated region of N. tabacum alcohol dehydrogenase gene (NtADH 5′-UTR) under the control of cauliflower mosaic virus (CaMV 35S) promoter was employed to improve the tENDO1 protein yield. To ease the purification process, tENDO1 was secreted into the culture medium and isolated using nickel affinity chromatography. The tENDO1 was estimated to be stably produced in an average of 0.7–0.9 % total soluble protein. Functional test on tENDO1 for mismatch detection demonstrated that tENDO1 retained mismatch specific endonuclease activity resembles its native protein. Further biochemical analysis showed that tENDO1 exhibited mismatch detection specificity and efficiency comparable to other commonly used endonucleases.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5



Enzyme-linked immunosorbent assay


Linsmaier and Skoog


5-Untranslated region of Nicotiana tabacum alcohol dehydrogenase gene


Truncated ENDO1


Targeting Induced Local Lesions IN Genomes


  • Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S (2002) Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18(2):298–305

    PubMed  Article  CAS  Google Scholar 

  • Geelan DNV, Inze DG (2001) A bright future for the bright yellow-2 cell culture. Plant Physiol 127:1375–1379

    Article  Google Scholar 

  • Girard LS, Bastin M, Courtois D (2004) Expression of the human milk protein sCD14 in tobacco plant cell culture. Plant Cell Tissue Organ Cult 78:253–260

    Article  CAS  Google Scholar 

  • Holsters M, Dewaele D, Depicker A, Messens E, Vanmontagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Mol Gen Genet 163:181–187

    PubMed  Article  CAS  Google Scholar 

  • Huang TK, McDonald KA (2009) Bioreactor engineering for recombinant protein production in plant cell suspension cultures. Biochem Eng J 45:168–184

    Article  CAS  Google Scholar 

  • Lai KS, Abdullah P, Yusoff K, Mahmood M (2011a) An efficient protocol for particle bombardment-mediated transformation of Centella asiatica callus. Acta Physiol Plant 33:2547–2552

    Article  Google Scholar 

  • Lai KS, Yusoff K, Mahmood M (2011b) Extracellular matrix as the early structural marker for Centella asiatica embryogenic tissues. Biol Plantarum 55(3):549–553

    Article  CAS  Google Scholar 

  • Lai KS, Kathoien-Nakayama P, Iwano M, Takayama S (2012a) A TILLING resource for functional genomics in Arabidopsis thaliana accession C24. Genes Genet Syst 87:291–297

    PubMed  CAS  Google Scholar 

  • Lai KS, Yusoff K, Mahmood M (2012b) Heterologous expression of hemagglutinin-neuraminidase protein from newcastle disease virus strain af2240 in Centella asiatica. Acta Biol Crac Ser Bot 54(1):1–6

    Google Scholar 

  • Lai KS, Yusoff K, Mahmood M (2013) Functional ectodomain of the hemagglutinin-neuraminidase protein is expressed in transgenic tobacco cells as a candidate vaccine against Newcastle disease virus. Plant Cell Tissue Organ Cult 112:117–121

    Article  CAS  Google Scholar 

  • Lee JM, Magnuson NS, An G, Reeves R (2000) Production of secreted foreign polypeptides in plant cell culture. United States Patent 6020169

  • Matsui T, Hori M, Shizawa N, Nakayama H, Shinmyo A, Yoshida K (2006) High-efficiency secretory production of peroxidase C1a using vascular transport engineering in transgenic tobacco. J Biosci Bioeng 102:102–109

    PubMed  Article  CAS  Google Scholar 

  • Minoia S, Petrozza A, D’Onofrio O, Piron F, Mosca G, Sozio G, Cellini F, Bendahmane A, Carriero F (2010) A new mutant genetic resource for tomato crop improvement by TILLING technology. BMC Res Notes 3:69

    PubMed  Article  Google Scholar 

  • Nakayama H, Yoshida K, Ono H, Murooka Y, Shinmyo A (2000) Ectoine, the compatible solute of Halomonas elongata, confers hyperosmotic tolerance in cultured tobacco cells. Plant Physiol 122:1239–1246

    PubMed  Article  CAS  Google Scholar 

  • Perez-Amador MA, Abler ML, De Rocher EJ, Thompson DM, van Hoof A, LeBrasseur ND, Lers A, Green PJ (2000) Identification of BFN1, a bifunctional nuclease induced during leaf and stem senescence in Arabidopsis. Plant Physiol 122:169–180

    PubMed  Article  CAS  Google Scholar 

  • Pires AS, Rosa S, Castanheira S, Fevereiro P, Abranches R (2012) Expression of a recombinant human erythropoietin in suspension cell cultures of Arabidopsis, tobacco and Medicago. Plant Cell Tissue Organ Cult 110:171–181

    Article  CAS  Google Scholar 

  • Qiao YS, Ling WD, Lomonossoff GP, Sun YB, Luo M, Li CQ, Jiang L, Xu ZF (2011) Improved expression and purification of recombinant human serum albumin from transgenic tobacco suspension culture. J Biotechnol 155:164–172

    Article  Google Scholar 

  • Satoh J, Kato K, Shinmyo A (2004) The 5′-untranslated region of the tobacco alcohol dehydrogenase gene functions as an effective translational enhancer in plant. J Biosci Bioeng 98:1–8

    PubMed  CAS  Google Scholar 

  • Sharp JM, Doran PM (2001a) Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol Bioeng 73(5):338–346

    PubMed  Article  CAS  Google Scholar 

  • Sharp JM, Doran PM (2001b) Strategies for enhancing monoclonal antibody accumulation in plant cell and organ cultures. Biotechnol Prog 17(6):979–992

    PubMed  Article  CAS  Google Scholar 

  • Suen PK, Shen J, Sun SSM, Jiang L (2010) Expression and characterization of two functional vacuolar sorting receptor (VSR) proteins, BP-80 and AtVSR4 from culture media of transgenic tobacco BY-2 cells. Plant Sci 179:68–76

    Article  CAS  Google Scholar 

  • Sunil Kumar GB, Ganapathi TR, Srinivas L, Revathi CJ, Bapat VA (2005) Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures. Biotecnol Lett 27:927–932

    Article  Google Scholar 

  • Triques K, Sturbois B, Gallais S, Dalmais M, Chauvun S, Clepet C, Aubourg S, Rameau C, Caboche M, Bendahmane A (2006) Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea. Plant J 51:1116–1125

    Article  Google Scholar 

  • Triques K, Piednoir E, Dalmais M, Schmidt J, Le Signor C, Sharkey M, Caboche M, Sturbois B, Bendahmane A (2008) Mutation detection using ENDO1: Application to disease diagnostics in humans and TILLING and Eco-TILLING in plants. BMC Mol Biol 9:42

    PubMed  Article  Google Scholar 

  • Xu JF, Kieliszewski M (2011) Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site. J Biotechnol 154:54–59

    PubMed  Article  CAS  Google Scholar 

  • Xu JF, Okada S, Tan L, Goodrum KJ, Kopchick JJ, Kieliszewski MJ (2010) Human growth hormone expressed in tobacco cells as an arabinogalactan-protein fusion glycoprotein has a prolonged serum life. Transgenic Res 19(5):849–867

    PubMed  Article  CAS  Google Scholar 

  • Yano A, Takekoshi M, Morita E, Imai S, Nisizawa T, Hanada N (2006) Production of fab fragment corresponding to surface protein antigen of Streptococcus mutants serotype c-derived peptide by Escherichia coli cultured tobacco cells. J Biosci Bioeng 101(3):251–256

    PubMed  Article  CAS  Google Scholar 

Download references


The authors are thankful to the members of Intercellular Communications Laboratory of Nara Institute of Science and Technology, Japan for their constructive suggestions and technical support on the study.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Kok Song Lai.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 11 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lai, K.S., Yusoff, K. Production of tENDO1 in stably transformed tobacco cell cultures for mismatch detection. Plant Cell Tiss Organ Cult 114, 287–294 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Mismatch detection
  • Plant cell culture
  • Secretion
  • tENDO1