Skip to main content
SpringerLink
Log in

Arabidopsis thaliana hairy roots for the production of heterologous proteins

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

To evaluate the ability of Arabidopsis thaliana hairy roots to produce heterologous proteins, hypocotyls were transformed with Rhizobium rhizogenes harbouring a green fluorescent protein gene (gfp) fused to a plant signal peptide sequence. Hairy root transgenic lines were generated from wild-type or mutant genotypes. A line secreted GFP at 130 mg/l of culture medium. Unlike as was previously found with turnip hairy roots, a His-tag was still attached to approximately 50 % of the protein. Control of the pH and addition of a protease inhibitor to the culture medium resulted in up to 87 % of the GFP retaining the His-tag. A. thaliana hairy roots expressing the human serpina1 (α-1-antitrypsin) gene secreted the protein, which was visible on a PAGE gel. Protein activity in the culture medium was demonstrated using an elastase inhibition assay. A. thaliana hairy roots can now be considered for the production of heterologous proteins, making it possible to mine the numerous genetic resources for enhancing protein production and quality.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Aird ELN, Hamill JD, Rhodes MJC (1988) Cytogenetic analysis of hairy root cultures from a number of plant species transformed by Agrobacterium rhizogenes. Plant Cell Tissue Organ Cult l5:47–57

    Article  Google Scholar 

  • Bandaranayake AD, Almo SC (2014) Recent advances in mammalian protein production. FEBS Lett 588:253–260

    Article  CAS  PubMed  Google Scholar 

  • Banerjee S, Singh S, Rahman LU (2012) Biotransformation studies using hairy root cultures—a review. Biotechnol Adv 30:461–468

    Article  CAS  PubMed  Google Scholar 

  • Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D (2008) Preventing unintended proteolysis in plant protein biofactories. Plant Biotechnol J 6:633–648

    Article  CAS  PubMed  Google Scholar 

  • Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, Murray E, Morck D, Moloney MM (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8:588–606

    Article  CAS  PubMed  Google Scholar 

  • Butaye KMJ, Goderis IJWM, Wouters PFJ, Pues JMTG, Delauré SL, Broekaert WF, Depicker A, Cammue BPA, De Bolle MFC (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 39:440–449

    Article  CAS  PubMed  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 

  • Chandran RP, Potty VP (2008) Induction of hairy roots through the mediation of four strains of Agrobacterium rhizogenes. Ind. J Biotechnol 7:122–128

    CAS  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 

  • De Wilde C, Van Houdt H, De Buck S, Angenon G, De Jaeger G, Depicker A (2000) Plants as bioreactors for protein production: avoiding the problem of transgene silencing. Plant Mol Biol 43:347–359

    Article  PubMed  Google Scholar 

  • Doran PM (2006) Foreign protein degradation and instability in plants and plant tissue cultures. Trends Biotechnol 24:436–432

    Article  Google Scholar 

  • Elmayan T, Balzergue S, Béon F, Bourdon V, Daubremet J, Guénet Y, Mourrain P, Palauqui JC, Vernhettes S, Vialle T, Wostrikoff K, Vaucheret H (1998) Arabidopsis mutants impaired in cosuppression. Plant Cell 10:1747–1757

    Article  CAS  PubMed  PubMed Central  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

    Article  CAS  PubMed  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 

  • Huang TK, Plesha MA, Falk BW, Dandekar AM, McDonald KA (2009) Bioreactor strategies for improving production yield and functionality of a recombinant human protein in transgenic tobacco cell cultures. Biotechnol Bioeng 102:508–520

    Article  CAS  PubMed  Google Scholar 

  • Huang TK, Plesha MA, McDonald KA (2010) Semicontinuous bioreactor production of a recombinant human therapeutic protein using a chemically inducible viral amplicon expression system in transgenic plant cell suspension cultures. Biotechnol Bioeng 106:408–421

    CAS  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

    Article  CAS  PubMed  Google Scholar 

  • Jha S, Agarwal S, Sanyal I, Jain GK, Amla DV (2012) Differential subcellular targeting of recombinant human α1-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants. Plant Sci 196:53–66

    Article  CAS  PubMed  Google Scholar 

  • Jullian-Pawlicki N, Lequart-Pillon M, Huynh-Cong L, Lesur D, Cailleu D, Mesnard F, Laberche JC, Gontier E, Boitel-Conti M (2015) Arylnaphthalene and aryltetralin-type lignans in hairy root cultures of Linum perenne, and the stereochemistry of 6-methoxypodophyllotoxin and one diastereoisomer by HPLC-MS and NMR spectroscopy. Phytochem Anal 26:310–319

    Article  CAS  PubMed  Google Scholar 

  • Kang D, Gho YS, Suh M, Kang C (2002) Highly sensitive and fast protein detection with Coomassie brilliant blue in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Bull Korean Chem Soc 23:1511–1512

    Article  CAS  Google Scholar 

  • Karimi M, Van Montagu M, Gheysen G (1999) Hairy root production in Arabidopsis thaliana: cotransformation with a promoter-trap vector results in complex T-DNA integration patterns. Plant Cell Rep 19:133–142

    Article  CAS  Google Scholar 

  • Köhnlein T, Welte T (2008) Alpha-1 antitrypsin deficiency: Pathogenesis, clinical presentation, diagnosis, and treatment. Am J Med 121:3–9

    Article  PubMed  Google Scholar 

  • Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I (2006) Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots. Plant Physiol 141:1185–1193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lallemand J, Bouché F, Desiron C, Stautemas J, de Lemos Esteves F, Périlleux C, Tocquin P (2015) Extracellular peptidase hunting for improvement of protein production in plant cells and roots. Front Plant Sci 6(37)

  • Limpens E, Ramos J, Franken C, Raz V, Compaan B, Franssen H, Bisseling T, Geurts R (2004) RNA interference in Agrobacterium rhizogenes transformed roots of Arabidopsis and Medicago truncatula. J Exp Bot 55:983–992

    Article  CAS  PubMed  Google Scholar 

  • Ma JKC, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman RM (2005) Plant-derived pharmaceuticals—the road forward. Trends Plant Sci 10:580–585

    Article  CAS  PubMed  Google Scholar 

  • McDonald KA, Hong LM, Trombly DM, Xie Q, Jackman AP (2005) Production of human α-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnol Prog 21:728–734

    Article  CAS  PubMed  Google Scholar 

  • Mehrotra S, Srivastava V, Rahman LU, Kukreja AK (2015) Hairy root biotechnology—indicative timeline to understand missing links and future outlook. Protoplasma 252:1189–1201

    Article  CAS  PubMed  Google Scholar 

  • Miguel D, Marum L (2011) An epigenetic view of plant cells cultured in vitro: somaclonal variation and beyond. J Exp Bot 62:3713–3725

    Article  CAS  PubMed  Google Scholar 

  • Ono NN, Tian L (2011) The multiplicity of hairy root cultures: Prolific possibilities. Plant Sci 180:439–446

    Article  CAS  PubMed  Google Scholar 

  • Peragine A, Yoshikawa M, Wu G, Heidi L, Albrecht HL, Poethig RS (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev 18:2368–2379

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Provart NJ, Alonso J, Assmann SM et al (2016) 50 years of Arabidopsis research: highlights and future directions. New Phytol 209:921–944

    Article  CAS  PubMed  Google Scholar 

  • Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5(172)

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

    Article  CAS  PubMed  Google Scholar 

  • Terashima M, Murai Y, Kawamura M, Nakanishi S, Stoltz T, Chen L, Drohan W, Rodriguez RL, Katoh S (1999) Production of functional human α1-antitrypsin by plant cell culture. Appl Microbiol Biotechnol 52:516–523

    Article  CAS  PubMed  Google Scholar 

  • Terashima M, Ejiri Y, Hashikawa N, Yoshida H (2001) Utilization of an alternative carbon source for efficient production of human α1-antitrypsin by genetically engineered rice cell culture. Biotechnol Prog 17:403–406

    Article  CAS  PubMed  Google Scholar 

  • Trexler MM, McDonald KA, Jackman AP (2002) Bioreactor production of human α1-antitrypsin using metabolically regulated plant cell cultures. Biotechnol Prog 18:501–508

    Article  CAS  PubMed  Google Scholar 

  • Trexler MM, McDonald KA, Jackman AP (2005) A cyclical semicontinuous process for production of human α1-antitrypsin using metabolically induced plant cell suspension cultures. Biotechnol Prog 21:321–328

    Article  CAS  PubMed  Google Scholar 

  • Xu J, Dolan MC, Medrano G, Cramer CL, Weathers PJ (2012) Green factory: Plants as bioproduction platforms for recombinant proteins. Biotechnol Adv 30:1171–1184

    Article  CAS  PubMed  Google Scholar 

  • Yakhnin AV, Vinokurov LM, Surin AK, Alakhov YB (1998) Green fluorescent protein purification by organic extraction. Protein Express Purif 14:382–386

    Article  CAS  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

    Article  CAS  PubMed  Google Scholar 

  • Zhang L, Shi J, Jiang D, Stupak J, Ou J, Qiu Q, An N, Li J, Yang D (2012) Expression and characterization of recombinant human alpha-antitrypsin in transgenic rice seed. J Biotechnol 164:300–308

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Yoann Huet for the design of the AAT sequence, Laurent Coquet (Plateforme de Protéomique Pissaro, University of Rouen) for LC-MS-MS analysis, Solène Bassard for technical assistance and Carol Robins for corrections to the manuscript. NTPM is grateful to the Vietnamese government and the University of Science and Technology of Hanoi for a PhD fellowship.

Author contributions

NTPM adapted the Arabidopsis transformation and root culture techniques, did the protein assays and formatted the data, MBC designed the experiments and edited the manuscript and FG designed the experiments, did the cloning experiment and wrote the paper.

Author information

Authors and Affiliations

  1. Biologie des plantes et innovation (BIOPI), Université de Picardie Jules Verne, 33 rue St Leu, 80039, Amiens, France

    Nga T. P. Mai, Michèle Boitel-Conti & François Guerineau

Authors
  1. Nga T. P. Mai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michèle Boitel-Conti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. François Guerineau
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to François Guerineau.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 92 KB)

Supplementary material 2 (DOCX 11357 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mai, N.T.P., Boitel-Conti, M. & Guerineau, F. Arabidopsis thaliana hairy roots for the production of heterologous proteins. Plant Cell Tiss Organ Cult 127, 489–496 (2016). https://doi.org/10.1007/s11240-016-1073-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-016-1073-7

Keywords

Search

Navigation