Im“plant”ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation

  • Hiroyuki Kajiura
  • Kazuhito Fujiyama
Part of the Methods in Molecular Biology book series (MIMB, volume 1321)


Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

Key words

Glycosyltransferase assay β1,4-galactosyltransferase Nicotiana tabacum L. cv. Bright Yellow 2 Agrobacterium-mediated gene expression High-performance liquid chromatography PA-sugar chain 


  1. 1.
    Palacpac NQ, Yoshida S, Sakai H et al (1999) Stable expression of human β1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci U S A 96:4692–4697PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Bakker H, Rouwendal GJ, Karnoup AS et al (2006) An antibody produced in tobacco expressing a hybrid β-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes. Proc Natl Acad Sci U S A 103:7577–7582PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Castilho A, Pabst M, Leonard R et al (2008) Construction of a functional CMP-sialic acid biosynthesis pathway in Arabidopsis. Plant Physiol 147:331–339PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Frey AD, Karg SR, Kallio PT (2009) Expression of rat β(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation. Plant Biotechnol J 7:33–48PubMedCrossRefGoogle Scholar
  5. 5.
    Castilho A, Strasser R, Stadlmann J et al (2010) In planta protein sialylation through over-expression of the respective mammalian pathway. J Biol Chem 285:15923–15930PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Gomord V, Chamberlain P, Jefferis R et al (2005) Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol 23:559–565PubMedCrossRefGoogle Scholar
  7. 7.
    Wilson IB (2002) Glycosylation of proteins in plants and invertebrates. Curr Opin Struct Biol 12:569–577PubMedCrossRefGoogle Scholar
  8. 8.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  9. 9.
    Misaki R, Fujiyama K, Seki T (2006) Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Biochem Biophys Res Commun 339:1184–1189PubMedCrossRefGoogle Scholar
  10. 10.
    Kajiura H, Misaki R, Fujiyama K et al (2011) Stable coexpression of two human sialylation enzymes in plant suspension-cultured tobacco cells. J Biosci Bioeng 111:471–477PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Hiroyuki Kajiura
    • 1
  • Kazuhito Fujiyama
    • 1
  1. 1.The International Center for BiotechnologyOsaka UniversityOsakaJapan

Personalised recommendations