Arabidopsis Hairy Roots Producing High Level of Active Human Gastric Lipase

Abstract

Arabidopsis hairy roots were used to produce human gastric lipase. When treated with 2,4-D, the hairy roots developed into thick organs that produced more protein than untreated roots. This was first assessed using green fluorescent protein-producing root lines from which the protein diffused into the culture medium. When growing hairy roots which express the human gastric lipase gene, very little lipase was found in the medium. Incubating the roots in a low pH buffer resulted in lipase diffusion into the buffer, avoiding the need for grinding. The activity of the enzyme on 4-methylumbellireryl-oleate and on tributyrin was determined. Approximately 6000 units of enzyme were recovered per gram of root. The enzyme was also extracted from freeze-dried roots before and after a 2-month storage period at room temperature. This work demonstrates the relevance of Arabidopsis hairy roots for the production of human gastric lipase.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

hGL:

Human gastric lipase

MU:

4-Methylumbellirerone

MUO:

4-Methylumbellireryl-oleate

References

  1. 1.

    Hellwig, S., Drossard, J., Twyman, R. M., & Fischer, R. (2004). Plant cell cultures for the production of recombinant proteins. Nature Biotechnology, 22, 1415–1422.

    CAS  PubMed  Google Scholar 

  2. 2.

    Stoger, E., Ma, J. K. C., Fischer, R., & Christou, P. (2005). Sowing the seeds of success: Pharmaceutical proteins from plants. Current Opinion in Biotechnology, 16, 167–173.

    CAS  PubMed  Google Scholar 

  3. 3.

    Xu, J., Dolan, M. C., Medrano, G., Cramer, C. L., & Weathers, P. J. (2012). Green factory: Plants as bioproduction platforms for recombinant proteins. Biotechnology Advances, 30, 1171–1184.

    CAS  PubMed  Google Scholar 

  4. 4.

    Ma, J. K. C., Drossard, J., Lewis, D., Altmann, F., Boyle, J., Christou, P., et al. (2015). Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnology Journal, 13, 1106–1120.

    CAS  PubMed  Google Scholar 

  5. 5.

    D’Aoust, M. A., Couture, M. M. J., Charland, N., Trépanier, S., Landry, N., Ors, F., et al. (2010). The production of hemagglutinin-based virus-like particles in plants: A rapid, efficient and safe response to pandemic influenza. Plant Biotechnology Journal, 8, 607–619.

    PubMed  Google Scholar 

  6. 6.

    Sainsbury, F., & Lomonossoff, G. P. (2014). Transient expressions of synthetic biology in plants. Current Opinion in Plant Biology, 19, 1–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7.

    Fujiuchi, N., Matoba, N., & Matsuda, R. (2016). Environment control to improve recombinant protein yields in plants based on Agrobacterium-mediated transient gene expression. Frontiers in Bioengineering and Biotechnology, 4, 23.

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Gaume, A., Komarnytsky, S., Borisjuk, N., & Raskin, I. (2003). Rhizosecretion of recombinant proteins from plant hairy roots. Plant Cell Reports, 21, 1188–1193.

    CAS  PubMed  Google Scholar 

  9. 9.

    Sabalza, M., Christou, P., & Capell, T. (2014). Recombinant plant-derived pharmaceutical proteins: current technical and economic bottlenecks. Biotechnology Letters, 36, 2367–2379.

    CAS  PubMed  Google Scholar 

  10. 10.

    Maxmen, A. (2012). Drug-making plant blooms. Nature, 485, 160.

    CAS  PubMed  Google Scholar 

  11. 11.

    Shaaltiel, Y., Gingis-Velitski, S., Tzaban, S., Fiks, N., Tekoah, Y., & Aviezer, D. (2015). Plant-based oral delivery of ß-glucocerebrosidase as an enzyme replacement therapy for Gaucher’s disease. Plant Biotechnology Journal, 13, 1033–1040.

    CAS  PubMed  Google Scholar 

  12. 12.

    Sack, M., Hofbauer, A., Fischer, R., & Stoger, E. (2015). The increasing value of plant-made proteins. Current Opinion in Biotechnology, 32, 163–170.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Yamamoto, T., Hoshikawa, K., Ezura, K., Okazawa, R., Fujita, S., Takaoka, M., et al. (2018). Improvement of the transient expression system for production of recombinant proteins in plants. Scientific Reports, 8, 4755.

    PubMed  PubMed Central  Google Scholar 

  14. 14.

    Georgiev, M. I., Agostini, E., Ludwig-Müller, J., & Xu, J. (2012). Genetically transformed roots: from plant disease to biotechnological resource. Trends in Biotechnology, 30, 528–537.

    CAS  PubMed  Google Scholar 

  15. 15.

    Aird, E. L. D., Hamill, J. D., & Rhodes, M. J. C. (1988). Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell, Tissue and Organ Culture, 15, 47–57.

    Google Scholar 

  16. 16.

    Häkkinen, S. T., Moyano, E., Cusido, R. M., & Oksman-Caldentey, K. M. (2016). Exploring the metabolic stability of engineered hairy roots after 16 years maintenance. Frontiers in Plant Science, 7, 1486.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Sun, J., Ma, L., San, K. Y., & Peebles, C. A. M. (2017). Still stable after 11 years: A Catharanthus roseus hairy root line maintains inducible expression of anthranilate synthase. Biotechnology Progress, 33, 66–69.

    CAS  PubMed  Google Scholar 

  18. 18.

    Joh, L. D., Wroblewski, T., Ewing, N. N., & VanderGheynst, J. S. (2005). High-level transient expression of recombinant protein in lettuce. Biotechnology and Bioengineering, 91, 861–871.

    CAS  PubMed  Google Scholar 

  19. 19.

    Clarke, J. L., Paruch, L., Dobrica, M. O., Caras, I., Tucureanu, C., Onu, A., et al. (2017). Lettuce-produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination. Plant Biotechnology Journal, 15, 1611–1621.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., et al. (2007). Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system. Plant Biotechnology Journal, 5, 579–590.

    CAS  PubMed  Google Scholar 

  21. 21.

    Huet, Y., Ele Ekouna, J. P., Caron, A., Mezreb, K., Boitel-Conti, M., & Guerineau, F. (2014). Production and secretion of a heterologous protein by turnip hairy roots with superiority over tobacco hairy roots. Biotechnology Letters, 36, 181–190.

    CAS  PubMed  Google Scholar 

  22. 22.

    Cardon, F., Pallisse, R., Bardor, M., Caron, A., Vanier, J., Ele Ekouna, J. P., et al. (2019). Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-l-iduronidase. Plant Biotechnology Journal, 17, 505–516.

    CAS  PubMed  Google Scholar 

  23. 23.

    von Schaewen, A., Jeong, I. S., Rips, S., Fukudome, A., Tolley, J., Nagashima, Y., et al. (2018). Improved recombinant protein production in Arabidopsis thaliana. Plant Signaling & Behavior, 13, e1486149.

    Google Scholar 

  24. 24.

    Butaye, K. M. J., Goderis, I. J. W. M., Wouters, P. F. J., Pues, J. M. T. G., Delauré, S. L., Broekaert, W. F., et al. (2004). Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. The Plant Journal, 39, 440–449.

    CAS  PubMed  Google Scholar 

  25. 25.

    Mai, N. T. P., Boitel-Conti, M., & Guerineau, F. (2016). Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell, Tissue and Organ Culture, 127, 489–496.

    CAS  Google Scholar 

  26. 26.

    Ele Ekouna, J. P., Boitel-Conti, M., Lerouge, P., Bardor, M., & Guerineau, F. (2017). Enhanced production of recombinant human gastric lipase in turnip hairy roots. Plant Cell, Tissue and Organ Culture, 131, 601–610.

    CAS  Google Scholar 

  27. 27.

    Lonoce, C., Marusic, C., Morrocchi, E., Salzano, A. M., Scaloni, A., Novelli, F., et al. (2019). Enhancing the secretion of a glyco-engineered anti-CD20 scFv-Fc antibody in hairy root cultures. Biotechnology Journal, 14, e1800081.

    PubMed  Google Scholar 

  28. 28.

    Aloulou, A., & Carrière, F. (2008). Gastric lipase: an extremophilic interfacial enzyme with medical applications. Cellular and Molecular Life Sciences, 65, 851–854.

    CAS  PubMed  Google Scholar 

  29. 29.

    Tschofen, M., Knopp, D., Hood, E., & Stöger, E. (2016). Plant molecular farming: Much more than medicines. Annual Review of Analytical Chemistry, 9, 271–294.

    PubMed  Google Scholar 

  30. 30.

    Peragine, A., Yoshikawa, M., Wu, G., Albrecht, H. L., & Poethig, R. S. (2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes & Development, 18, 2368–2379.

    CAS  Google Scholar 

  31. 31.

    Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248–254.

    CAS  PubMed  Google Scholar 

  32. 32.

    Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., et al. (1985). Measurement of protein using bicinchoninic acid. Analytical Biochemistry, 150, 76–85.

    CAS  PubMed  Google Scholar 

  33. 33.

    Gargouri, Y., Pieroni, G., Riviere, C., Sauniere, J. F., Lowe, P. A., Sarda, L., et al. (1986). Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions. Gastroenterology, 91, 919–925.

    CAS  PubMed  Google Scholar 

  34. 34.

    Kang, D., Gho, Y. S., Suh, M., & Kang, C. (2002). Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Bulletin of the Korean Chemical Society, 23, 1511–1512.

    CAS  Google Scholar 

  35. 35.

    Atta, R., Laurens, L., Boucheron-Dubuisson, E., Guivarch, A., Carnero, E., Giraudat-Pautot, V., et al. (2009). Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. The Plant Journal, 57, 626–644.

    CAS  PubMed  Google Scholar 

  36. 36.

    Smetana, O., Mäkilä, R., Lyu, M., Amiryousefi, A., Sanchez Rodríguez, F., Wu, M. F., et al. (2019). High levels of auxin signalling define the stem-cell organizer of the vascular cambium. Nature, 565, 485–489.

    CAS  PubMed  Google Scholar 

  37. 37.

    Sams, L., Amara, S., Chakroun, A., Coudre, S., Paume, J., Giallo, J., et al. (2017). Constitutive expression of human gastric lipase in Pichia pastoris and site-directed mutagenesis of key lid-stabilizing residues. Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids, 1862, 1025–1034.

    CAS  PubMed  Google Scholar 

  38. 38.

    Mokrzycki-Issartel, N., Bouchon, B., Farrer, S., Berland, P., Laparra, H., Madelmont, J. C., et al. (2003). A transient tobacco expression system coupled to MALDI-TOF-MS allows validation of the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein produced in plants. FEBS Letters, 552, 170–176.

    CAS  PubMed  Google Scholar 

  39. 39.

    Zhong, Q., Gu, Z., & Glatz, C. E. (2006). Extraction of recombinant dog gastric lipase from transgenic corn seed. Journal of Agriculture and Food Chemistry, 54, 8086–8092.

    CAS  Google Scholar 

  40. 40.

    Vardakou, M., Sainsbury, F., Rigby, N., Mulholland, F., & Lomonossoff, G. P. (2012). Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expression and Purification, 81, 69–74.

    CAS  PubMed  Google Scholar 

  41. 41.

    Sapan, C. V., Lundblad, R. L., & Price, N. C. (1999). Colorimetric protein assay techniques. Biotechnology and Applied Biochemistry, 29, 99–108.

    CAS  PubMed  Google Scholar 

  42. 42.

    Sarmah, N., Revathi, D., Sheelu, G., Yamuna Rani, K., Sridhar, S., Mehtab, V., et al. (2018). Recent advances on sources and industrial applications of lipases. Biotechnology Progress, 34, 5–28.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Emmanuel Petit (BIOPI) for his help with titrimetric assays and Astrid Boitel and Keith Randall for editing the manuscript.

Funding

Funding was provided by French "ministère de lʼEnseignement supérieur, de la recherche et de lʼinnovation" (Grant No. EA3900).

Author information

Affiliations

Authors

Corresponding author

Correspondence to François Guerineau.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Guerineau, F., Mai, N.T.P. & Boitel-Conti, M. Arabidopsis Hairy Roots Producing High Level of Active Human Gastric Lipase. Mol Biotechnol 62, 168–176 (2020). https://doi.org/10.1007/s12033-019-00233-y

Download citation

Keywords

  • Auxin
  • 2,4-D
  • Arabidopsis
  • Gastric lipase
  • Hairy roots