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Enhanced production of recombinant human gastric lipase in turnip hairy roots

Abstract

Treatment with 2,4-D induced the development of callus-like structures on Brassica rapa hairy roots grown in liquid medium. The structures probably originated from pericycle cell divisions and developed in a non-concentric manner to give conical organs, which did not separate from the roots, whereas the root cortical and epidermal cell layers degenerated. Various cell types were found within the structures, indicating that these differ from simple calli. They are likely to represent secondary root primordia that fail to elongate as a result of hormonal pressure. Hairy roots transformed with a human gene encoding gastric lipase (hGL) were used to produce the recombinant protein. Lipase activity was measured in the culture medium of these roots. The analysis of N-glycans on hGL produced by hairy roots revealed the presence of paucimannosidic N-glycans. Moreover, when the root cultures were treated with 2,4-D, approximately 2.7 times more lipase activity was measured in the culture medium, compared to untreated root cultures, revealing the higher efficiency of this system for the production of a heterologous protein. In addition, some His-tag epitope was found on EGFP in the medium of 2,4-D-treated roots expressing a his-egfp gene, whereas the same tag was totally degraded in the medium of untreated roots, suggesting a reducing effect of 2,4-D on the release of proteolytic activity in the culture medium. We propose to name this new hairy root-derived protein production process “rhizocalli”.

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References

  • Aloulou A, Carrière F (2008) Gastric lipase: an extremophilic interfacial enzyme with medical applications. Cell Mol Life Sci 65:851–854

    CAS  Article  PubMed  Google Scholar 

  • Baiet B, Burel C, Saint-Jean B, Louvet R, Menu-Bouaouiche L, Kiefer-Meyer M-C, Mathieu-Rivet E, Lefebvre T, Castel H, Carlier A, Cadoret J-P, Lerouge P, Bardor M (2011) N-glycans of Phaeodactylum tricornutum diatom and functional characterization of its N-acetylglucosaminyltransferase I enzyme. J Biol Chem 286:6152–6164

    CAS  Article  PubMed  Google Scholar 

  • Bakker H, Bardor M, Molhoff J, Gomord V, Elbers I, Stevens L, Jordi W, Lommen A, Faye L, Lerouge P, Bosch D (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci USA 98:2899–2904

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Campanoni P, Nick P (2005) Auxin-dependent cell division and cell elongation. 1-Naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid activate different pathways. Plant Physiol 137:939–948

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Castilho A, Windwarder M, Gattinger P, Mach L, Strasser R, Altmann F, Steinkellner H (2014) Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiol 166:1839–1851

    Article  PubMed  PubMed Central  Google Scholar 

  • Crabbe T, Weir AN, Walton EF, Brown ME, Sutton CW, Tretout N, Bonnerjea J, Lowe PA, Yarranton GT (1996) The secretion of active recombinant human gastric lipase by Saccharomyces cerevisiae. Protein Expr Purif 7:229–236

    CAS  Article  PubMed  Google Scholar 

  • Datla RSS, Hammerlindl JK, Panchuk B, Pelcher LE, Keller W (1992) Modified binary plant transformation vectors with the wild-type gene encoding NPTII. Gene 211:383–384

    Google Scholar 

  • Gargouri Y, Pieroni G, Riviere C, Sauniere JF, Lowe PA, Sarda L, Verger R (1986) Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions. Gastroenterology 91:919–925

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  • Georgiev MI, Agostini E, Ludwig-Müller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotechnol 30:528–537

    CAS  Article  PubMed  Google Scholar 

  • Gruber V, Berna PP, Arnaud T et al (2001) Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase. Mol Breed 7:329–340

    CAS  Article  Google Scholar 

  • Guerineau F (1995) Tools for expressing foreign genes in plants. Methods Mol Biol 49:1–32

    CAS  PubMed  Google Scholar 

  • Häkkinen ST, Raven N, Henquet M, Laukkanen ML, Anderlei T, Pitkänen JP, Twyman RM, Bosch D, Oksman-Caldentey KM, Schillberg S, Ritala A (2014) Molecular farming in tobacco hairy roots by triggering the secretion of a pharmaceutical antibody. Biotechnol Bioeng 111:336–346

    Article  PubMed  Google Scholar 

  • Ho SCL, Bardor M, Feng H, Zhang M, Hosokai Y, Song Z, Tong YW, Yap MGS, Yang YS (2012) IRES-mediated tricistronic vectors for enhancing generation of high monoclonal antibody expressing CHO cell lines. J Biotechnol 157:130–139

    CAS  Article  PubMed  Google Scholar 

  • Huet Y, Ele Ekouna JP, 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. Biotechnol Lett 36:181–190

    CAS  Article  PubMed  Google Scholar 

  • Jacks TJ, Kircher HW (1967) Fluorometric assay for the hydrolytic activity of lipase using fatty acyl esters of 4-methylumbelliferone. Anal Biochem 21:279–285

    CAS  Article  PubMed  Google Scholar 

  • Liebminger E, Veit C, Pabst M, Batoux M, Zipfel C, Altmann F, Mach L, Strasser R (2011) β-N-acetylhexosaminidases HEXO1 and HEXO3 are responsible for the formation of paucimannosidic N-glycans in Arabidopsis thaliana. J Biol Chem 286:10793–10802

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Madeira LM, Szeto TH, Henquet M, Raven N, Runions J, Huddleston J, Garrard I, Drake PMW, Ma JKC (2016) High-yield production of a human monoclonal IgG by rhizosecretion in hydroponic tobacco cultures. Plant Biotechnol J 14:615–624

    CAS  Article  PubMed  Google Scholar 

  • Mathieu-Rivet E, Scholz M, Arias C, Dardelle F, Schulze S, Le Mauff F, Teo G, Hochmal AK, Blanco-Rivero A, Loutelier-Bourhis C, Kiefer-Meyer M-C, Fufezan C, Burel C, Lerouge P, Martinez F, Bardor M, Hippler M (2013) Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures. Mol Cell Proteomics 12:3160–3183

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Mokrzycki-Issartel N, Bouchon B, Farrer S, Berland P, Laparra H, Madelmont JC, Theisen M (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 Lett 552:170–176

    CAS  Article  PubMed  Google Scholar 

  • Roussel A, Canaan S, Egloff MP, Rivière M, Dupuis L, Verger R, Cambillau C (1999) Crystal structure of human gastric lipase and model of lysosomal acid lipase, two lipolytic enzymes of medical interest. J Biol Chem 274:16995–17002

    CAS  Article  PubMed  Google Scholar 

  • Roussel A, Miled N, Berti-Dupuis L, Rivière M, Spinelli S, Berna P, Gruber V, Verger R, Cambillau C (2002) Crystal structure of the open form of dog gastric lipase in complex with a phosphonate inhibitor. J Biol Chem 277:2266–2274

    CAS  Article  PubMed  Google Scholar 

  • Séveno M, Cabrera G, Triguero A, Burel C, Leprince J, Rihouey C, Vézina L-P, D’Aoust M-A, Rudd P, Royle L, Dwek R, Harvey J, Lerouge P, Cremata J, Bardor M (2008) Plant N-glycan profiling of minute amounts of material. Anal Biochem 379:66–72

    Article  PubMed  Google Scholar 

  • Shivaraj SM, Singh A (2016) Sequence variation in Brassica AP2 and analysis of interaction of AP2-miR172 regulatory module. Plant Cell Tissue Organ Cult 125:191–206

    CAS  Article  Google Scholar 

  • Smerdon GR, Aves SJ, Walton EF (1995) Production of human gastric lipase in the fission yeast Schizosaccharomyces pombe. Gene 165:313–318

    CAS  Article  PubMed  Google Scholar 

  • Spivak M, Weston J, Bottou L, Käll L, Noble WS (2009) Improvements to the Percolator algorithm for peptide identification from shotgun proteomics data sets. J Proteome Res 8:3737–3745

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Sugimoto K, Jiao Y, Meyerowitz EM (2010) Arabidopsis regeneration from multiple tissues occurs via a root development pathway. Dev Cell 18:463–471

    CAS  Article  PubMed  Google Scholar 

  • van Oberbeek J, Blondeau R, Horne V (1951) Difference in activity between 2,4-dichlorophenoxyacetic acid and other auxins, and its significance in herbicidal action. Plant Physiol 26:687–696

    Article  Google Scholar 

  • Vanier G, Hempel F, Chan P, Rodamer M, Vaudry D, Maier UG, Lerouge P, Bardor M (2015) Biochemical characterization of human anti-hepatitis B monoclonal antibody produced in the microalgae Phaeodactylum tricornutum. PLoS ONE 10:e0139282. doi: 10.1371/journal.pone.0139282

    Article  PubMed  PubMed Central  Google Scholar 

  • Vardakou M, Sainsbury F, Rigby N, Mulholland F, Lomonossoff GP (2012) Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system. Protein Expr Purif 81:69–74

    CAS  Article  PubMed  Google Scholar 

  • Wicker-Planquart C, Canaan S, Rivière M, Dupuis L, Verger R (1996) Expression in insect cells and purification of a catalytically active recombinant human gastric lipase. Protein Eng 9:1225–1232

    CAS  Article  PubMed  Google Scholar 

  • Wicker-Planquart C, Canaan S, Rivière M, Dupuis L (1999) Site-directed removal of N-glycosylation sites in human gastric lipase. Eur J Biochem 262:644–651

    CAS  Article  PubMed  Google Scholar 

  • Young JM, Kuykendall LD, Martinez-Romero E, Kerr A, Sawada H (2001) A revision of Rhizobium Frank 1889, with an emended description of the genus, and the inclusion of all species of Agrobacterium Conn 1942 and Allorhizobium undicola de Lajudie et al. 1998 as new combinations: Rhizobium radiobacter, R. rhizogenes, R. rubi, R. undicola and R. vitis. Int J Syst Evol Microbiol 51:89–103

    CAS  Article  PubMed  Google Scholar 

  • Zhong Q, Gu Z, Glatz CE (2006) Extraction of recombinant dog gastric lipase from transgenic corn seed. J Agric Food Chem 54:8086–8092

    CAS  Article  PubMed  Google Scholar 

  • Zimmer M (2002) Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev 102:759–781

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Jean-William Dupuy (Plateforme Protéome de Bordeaux) for the nano-LC-MS/MS analysis, Solène Bassard, Gaëtan Vanier and Philippe Chan (PISSARO proteomic platform, Université de Rouen Normandie) for technical assistance, Carol Robins for corrections to the manuscript, and the European Regional Development Fund (FEDER), the “Région Picardie”, and the Root Lines Technology company for funding part of this project.

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Authors and Affiliations

Authors

Contributions

All authors contributed to the experiment design. JPEE did the transformation experiment, the lipase PAGE and Western analyses, the lipase assays, analysed and formatted the data and reviewed and edited the manuscript. MB performed the N-glycan analysis, wrote the relevant part of the M&M section, and reviewed and edited the manuscript. MBC initiated the hairy root work and reviewed and edited the manuscript. PL reviewed and edited the manuscript. FG initiated the rhizocalli work, did the cloning and the microscopy experiments, the GFP assay and Western analysis, formatted the data and wrote the paper.

Corresponding author

Correspondence to François Guerineau.

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Conflict of interest

JPEE, MBC and FG are inventors on patents EP2385130. Method for producing recombinant proteins from plant hairy roots and WO 2016/185122 A1. Method for producing proteins from a plant structure owned by the University of Picardie Jules Verne. MBC is a founder, shareholder and scientific adviser of the company Root Lines Technology which holds the patent licences.

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Communicated by Yi Li.

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Ele Ekouna, JP., Boitel-Conti, M., Lerouge, P. et al. Enhanced production of recombinant human gastric lipase in turnip hairy roots. Plant Cell Tiss Organ Cult 131, 601–610 (2017). https://doi.org/10.1007/s11240-017-1309-1

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  • DOI: https://doi.org/10.1007/s11240-017-1309-1

Keywords

  • Auxin
  • Brassica
  • Gastric lipase
  • N-Glycosylation
  • Hairy roots