Skip to main content
SpringerLink
Log in

Glycoprotein production in moss bioreactors

  • Review
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Complex multimeric recombinant proteins such as therapeutic antibodies require a eukaryotic expression system. Transgenic plants may serve as promising alternatives to the currently favored mammalian cell lines or hybridomas. In contrast to prokaryotic systems, posttranslational modifications of plant and human proteins resemble each other largely, among those, protein N-glycosylation of the complex type. However, a few plant-specific sugar residues may cause immune reactions in humans, representing an obstacle for the broad use of plant-based systems as biopharmaceutical production hosts. The moss Physcomitrella patens represents a flexible tissue-culture system for the contained production and secretion of recombinant biopharmaceuticals in photobioreactors. The recent synthesis of therapeutic proteins as a scFv antibody fragment or the large and heavily modified complement regulator factor H demonstrate the versatility of this expression system. A uniquely efficient gene targeting mechanism can be employed to precisely engineer the glycosylation machinery for recombinant products. In this way, P. patens lines with non-immunogenic optimized glycan structures were created. Therapeutic antibodies produced in these strains exhibited antibody-dependent cellular cytotoxicity superior to the same molecules synthesized in mammalian cell lines.

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

Similar content being viewed by others

References

  • Altmann F (2007) The role of protein glycosylation in allergy. Int Arch Allergy Immunol 142:99–115

    Article  PubMed  CAS  Google Scholar 

  • Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, 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

    Google Scholar 

  • Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P (2003) Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1, 3)-fucose and core xylose. Glycobiology 13:427–434

    Article  PubMed  CAS  Google Scholar 

  • Baur A, Reski R, Gorr G (2005) Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens. Plant Biotechnol J 3:331–340

    Article  PubMed  CAS  Google Scholar 

  • Beck A, Wagner-Rousset E, Bussat MC, Lokteff M, Klinguer-Hamour C, Haeuw JF, Goetsch L, Wurch T, Van Dorsselaer A, Corvaia N (2008) Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins. Curr Pharm Biotechnol 9:482–501

    Article  PubMed  CAS  Google Scholar 

  • Bencurova M, Hemmer W, Focke-Tejkl M, Wilson IB, Altmann F (2004) Specificity of IgG and IgE antibodies against plant and insect glycoprotein glycans determined with artificial glycoforms of human transferrin. Glycobiology 14:457–466

    Article  PubMed  CAS  Google Scholar 

  • Buttner-Mainik A, Parsons J, Jerome H, Hartmann A, Lamer S, Schaaf A, Schlosser A, Zipfel PF, Reski R, Decker EL (2011) Production of biologically active recombinant human factor H in Physcomitrella. Plant Biotechnol J 9:373–383

    Article  PubMed  Google Scholar 

  • Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P, Kunert R, Quendler H, Pabst M, Leonard R, Altmann F, Steinkellner H (2010) In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 285:15923–15930

    Article  PubMed  CAS  Google Scholar 

  • Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24:1591–1597

    Article  PubMed  CAS  Google Scholar 

  • Decker EL, Reski R (2007) Moss bioreactors producing improved biopharmaceuticals. Curr Opin Biotechnol 18:393–398

    Article  PubMed  CAS  Google Scholar 

  • Decker EL, Reski R (2008) Current achievements in the production of complex biopharmaceuticals with moss bioreactors. Bioproc Biosyst Eng 31:3–9

    Article  CAS  Google Scholar 

  • Durocher Y, Butler M (2009) Expression systems for therapeutic glycoprotein production. Curr Opin Biotechnol 20:700–707

    Article  PubMed  CAS  Google Scholar 

  • Erbayraktar S, Grasso G, Sfacteria A, Xie QW, Coleman T, Kreilgaard M, Torup L, Sager T, Erbayraktar Z, Gokmen N, Yilmaz O, Ghezzi P, Villa P, Fratelli M, Casagrande S, Leist M, Helboe L, Gerwein J, Christensen S, Geist MA, Pedersen LO, Cerami-Hand C, Wuerth JP, Cerami A, Brines M (2003) Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity in vivo. Proc Natl Acad Sci USA 100:6741–6746

    Article  PubMed  CAS  Google Scholar 

  • Faye L, Gomord V (2010) Success stories in molecular farming-a brief overview. Plant Biotechnol J 8:525–528

    Article  PubMed  Google Scholar 

  • Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM (2004) Plant-based production of biopharmaceuticals. Curr Opin Plant Biol 7:152–158

    Article  PubMed  CAS  Google Scholar 

  • Frank W, Decker EL, Reski R (2005) Molecular tools to study Physcomitrella patens. Plant Biol 7:220–227

    Article  PubMed  CAS  Google Scholar 

  • Garcia-Casado G, Sanchez-Monge R, Chrispeels MJ, Armentia A, Salcedo G, Gomez L (1996) Role of complex asparagine-linked glycans in the allergenicity of plant glycoproteins. Glycobiol 6:471–477

    Article  CAS  Google Scholar 

  • Gitzinger M, Parsons J, Reski R, Fussenegger M (2009) Functional cross-kingdom conservation of mammalian and moss (Physcomitrella patens) transcription, translation and secretion machineries. Plant Biotechnol J 7:73–86

    Article  PubMed  CAS  Google Scholar 

  • Gomord V, Chamberlain P, Jefferis R, Faye L (2005) Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol 23:559–565

    Article  PubMed  CAS  Google Scholar 

  • Gomord V, Fitchette AC, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud D, Faye L (2010) Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol J 8:564–587

    Article  PubMed  CAS  Google Scholar 

  • Gorr G, Jost W (2005) Glycosylation design in transgenic moss for better product efficacy. Bioprocess J 4:26–30

    Google Scholar 

  • Gremillon L, Kiessling J, Hause B, Decker EL, Reski R, Sarnighausen E (2007) Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens. New Phytol 176:299–310

    Article  PubMed  Google Scholar 

  • Hellwig S, Drossard J, Twyman RM, Fischer R (2004) Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22:1415–1422

    Article  PubMed  CAS  Google Scholar 

  • Hohe A, Decker EL, Gorr G, Schween G, Reski R (2002) Tight control of growth and cell differentiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor cultures. Plant Cell Rep 20:1135–1140

    Article  CAS  Google Scholar 

  • Horstmann V, Huether CM, Jost W, Reski R, Decker EL (2004) Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol 4:13

    Article  PubMed  Google Scholar 

  • Huether CM, Lienhart O, Baur A, Stemmer C, Gorr G, Reski R, Decker EL (2005) Glyco-engineering of moss lacking plant-specific sugar residues. Plant Biol 7:292–299

    Article  PubMed  CAS  Google Scholar 

  • Jin C, Altmann F, Strasser R, Mach L, Schahs M, Kunert R, Rademacher T, Glossl J, Steinkellner H (2008) A plant-derived human monoclonal antibody induces an anti-carbohydrate immune response in rabbits. Glycobiology 18:235–241

    Article  PubMed  CAS  Google Scholar 

  • Jost W, Link S, Horstmann V, Decker EL, Reski R, Gorr G (2005) Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression. Curr Genet 47:111–120

    Article  PubMed  CAS  Google Scholar 

  • Karg SR, Kallio PT (2009) The production of biopharmaceuticals in plant systems. Biotechnol Adv 27:879–894

    Article  PubMed  CAS  Google Scholar 

  • Khraiwesh B, Arif MA, Seumel GI, Ossowski S, Weigel D, Reski R, Frank W (2010) Transcriptional control of gene expression by microRNAs. Cell 140:111–122

    Article  PubMed  CAS  Google Scholar 

  • Kiessling J, Kruse S, Rensing SA, Harter K, Decker EL, Reski R (2000) Visualization of a cytoskeleton-like FtsZ network in chloroplasts. J Cell Biol 151:945–950

    Article  PubMed  CAS  Google Scholar 

  • Kiessling J, Martin A, Gremillon L, Rensing SA, Nick P, Sarnighausen E, Decker EL, Reski R (2004) Dual targeting of plastid division protein FtsZ to chloroplasts and the cytoplasm. EMBO Rep 5:889–894

    Article  PubMed  CAS  Google Scholar 

  • Koprivova A, Altmann F, Gorr G, Kopriva S, Reski R, Decker EL (2003) N-Glycosylation in the moss Physcomitrella patens is organized similarly to higher plants. Plant Biol 5:582–591

    Article  CAS  Google Scholar 

  • Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, Reski R, Decker EL (2004) Targeted knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant Biotechnol J 2:517–523

    Article  PubMed  CAS  Google Scholar 

  • Lang D, Eisinger J, Reski R, Rensing SA (2005) Representation and high-quality annotation of the Physcomitrella patens transcriptome demonstrates a high proportion of proteins involved in metabolism in mosses. Plant Biol 7:238–250

    Article  PubMed  CAS  Google Scholar 

  • Lerouge P, Cabanes-Macheteau M, Rayon C, Fischette-Laine AC, Gomord V, Faye L (1998) N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38:31–48

    Article  PubMed  CAS  Google Scholar 

  • Lucumi A, Posten C (2006) Establishment of long-term perfusion cultures of recombinant moss in a pilot tubular photobioreactor. Proc Biochem 41:2180–2187

    Article  CAS  Google Scholar 

  • Lucumi A, Posten C, Pons MN (2005) Image analysis supported moss cell disruption in photo-bioreactors. Plant Biol 7:276–282

    Article  PubMed  CAS  Google Scholar 

  • Mari A (2002) IgE to cross-reactive carbohydrate determinants: analysis of the distribution and appraisal of the in vivo and in vitro reactivity. Int Arch Allergy Immunol 129:286–295

    Article  PubMed  CAS  Google Scholar 

  • Mosquna A, Katz A, Decker EL, Rensing SA, Reski R, Ohad N (2009) Regulation of stem cell maintenance by the Polycomb protein FIE has been conserved during land plant evolution. Development 136:2433–2444

    Article  PubMed  CAS  Google Scholar 

  • Nechansky A, Schuster M, Jost W, Siegl P, Wiederkum S, Gorr G, Kircheis R (2007) Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular cytotoxicity by glyco-engineering of therapeutic antibodies. Mol Immunol 44:1815–1817

    Article  PubMed  CAS  Google Scholar 

  • Nishiyama T, Fujita T, Shin IT, Seki M, Nishide H, Uchiyama I, Kamiya A, Carninci P, Hayashizaki Y, Shinozaki K, Kohara Y, Hasebe M (2003) Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution. Proc Natl Acad Sci USA 100:8007–8012

    Article  PubMed  CAS  Google Scholar 

  • Palacpac NQ, Yoshida S, Sakai H, Kimura Y, Fujiyama K, Yoshida T, Seki T (1999) Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci USA 96:4692–4697

    Google Scholar 

  • Perner-Nochta I, Lucumi A, Posten C (2007) Photoautotrophic cell and tissue culture in a tubular photobioreactor. Eng Life Sci 7:127–135

    Article  CAS  Google Scholar 

  • Preithner S, Elm S, Lippold S, Locher M, Wolf A, da Silva AJ, Baeuerle PA, Prang NS (2006) High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G. Mol Immunol 43:1183–1193

    Article  PubMed  CAS  Google Scholar 

  • Rademacher TW, Parekh RB, Dwek RA (1988) Glycobiology. Annu Rev Biochem 57:785–838

    Article  PubMed  CAS  Google Scholar 

  • Rensing SA, Lang D, Zimmer AD, Terry A, Salamov A, Shapiro H, Nishiyama T, Perroud PF, Lindquist EA, Kamisugi Y, Tanahashi T, Sakakibara K, Fujita T, Oishi K, Shin IT, Kuroki Y, Toyoda A, Suzuki Y, Hashimoto S, Yamaguchi K, Sugano S, Kohara Y, Fujiyama A, Anterola A, Aoki S, Ashton N, Barbazuk WB, Barker E, Bennetzen JL, Blankenship R, Cho SH, Dutcher SK, Estelle M, Fawcett JA, Gundlach H, Hanada K, Heyl A, Hicks KA, Hughes J, Lohr M, Mayer K, Melkozernov A, Murata T, Nelson DR, Pils B, Prigge M, Reiss B, Renner T, Rombauts S, Rushton PJ, Sanderfoot A, Schween G, Shiu SH, Stueber K, Theodoulou FL, Tu H, Van de Peer Y, Verrier PJ, Waters E, Wood A, Yang L, Cove D, Cuming AC, Hasebe M, Lucas S, Mishler BD, Reski R, Grigoriev IV, Quatrano RS, Boore JL (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science 319:64–69

    Article  PubMed  CAS  Google Scholar 

  • Reski R (1998) Development, genetics and molecular biology of mosses. Botanica Acta 111:1–15

    CAS  Google Scholar 

  • Richter U, Kiessling J, Hedtke B, Decker E, Reski R, Borner T, Weihe A (2002) Two RpoT genes of Physcomitrella patens encode phage-type RNA polymerases with dual targeting to mitochondria and plastids. Gene 290:95–105

    Article  PubMed  CAS  Google Scholar 

  • Schaaf A, Reski R, Decker EL (2004) A novel aspartic proteinase is targeted to the secretory pathway and to the vacuole in the moss Physcomitrella patens. Eur J Cell Biol 83:145–152

    Article  PubMed  CAS  Google Scholar 

  • Schaaf A, Tintelnot S, Baur A, Reski R, Gorr G, Decker EL (2005) Use of endogenous signal sequences for transient production and efficient secretion by moss (Physcomitrella patens) cells. BMC Biotechnol 5:30

    Article  PubMed  Google Scholar 

  • Schaefer DG, Zryd JP (1997) Efficient gene targeting in the moss Physcomitrella patens. Plant J 11:1195–1206

    Article  PubMed  CAS  Google Scholar 

  • Schulte J, Reski R (2004) High throughput cryopreservation of 140,000 Physcomitrella patens mutants. Plant Biol 6:119–127

    Article  PubMed  CAS  Google Scholar 

  • Schuster M, Jost W, Mudde GC, Wiederkum S, Schwager C, Janzek E, Altmann F, Stadlmann J, Stemmer C, Gorr G (2007) In vivo glyco-engineered antibody with improved lytic potential produced by an innovative non-mammalian expression system. Biotechnol J 2:700–708

    Article  PubMed  CAS  Google Scholar 

  • Schween G, Hohe A, Koprivova A, Reski R (2003) Effects of nutrients, cell density and culture techniques on protoplast regeneration and early protonema development in a moss, Physcomitrella patens. J Plant Physiol 160:209–212

    Article  PubMed  CAS  Google Scholar 

  • Scolnik PA (2009) mAbs: a business perspective. MAbs 1:179–184

    Article  PubMed  Google Scholar 

  • Shields RL, Lai J, Keck R, O’Connell LY, Hong K, Meng YG, Weikert SH, Presta LG (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J Biol Chem 277:26733–26740

    Article  PubMed  CAS  Google Scholar 

  • Shinkawa T, Nakamura K, Yamane N, Shoji-Hosaka E, Kanda Y, Sakurada M, Uchida K, Anazawa H, Satoh M, Yamasaki M, Hanai N, Shitara K (2003) The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J Biol Chem 278:3466–3473

    Article  PubMed  CAS  Google Scholar 

  • Smidkova M, Hola M, Spryngar M, Angelis KJ (2010) Expresssion of recombinant anti-CD20 scFv antibody in moss Physcomitrella patens. MOSS2010. In: The 13th Annual MOSS International Conference T16

  • Sourrouille C, Marquet-Blouin E, D’Aoust MA, Kiefer-Meyer MC, Seveno M, Pagny-Salehabadi S, Bardor M, Durambur G, Lerouge P, Vezina L, Gomord V (2008) Down-regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol J 6:702–721

    Article  PubMed  CAS  Google Scholar 

  • Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1, 2-linked xylose and core alpha1, 3-linked fucose. FEBS Lett 561:132–136

    Article  PubMed  CAS  Google Scholar 

  • Strasser R, Stadlmann J, Schahs M, Stiegler G, Quendler H, Mach L, Glossl J, Weterings K, Pabst M, Steinkellner H (2008) Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J 6:392–402

    Article  PubMed  CAS  Google Scholar 

  • Strepp R, Scholz S, Kruse S, Speth V, Reski R (1998) Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin. Proc Natl Acad Sci USA 95:4368–4373

    Article  PubMed  CAS  Google Scholar 

  • van Ree R, Cabanes-Macheteau M, Akkerdaas J, Milazzo JP, Loutelier-Bourhis C, Rayon C, Villalba M, Koppelman S, Aalberse R, Rodriguez R, Faye L, Lerouge P (2000) Beta(1, 2)-xylose and alpha(1, 3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 275:11451–11458

    Article  PubMed  Google Scholar 

  • Vietor R, Loutelier-Bourhis C, Fitchette AC, Margerie P, Gonneau M, Faye L, Lerouge P (2003) Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants. Planta 218:269–275

    Article  PubMed  CAS  Google Scholar 

  • Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252

    Article  PubMed  CAS  Google Scholar 

  • Weise A, Rodriguez-Franco M, Timm B, Hermann M, Link S, Jost W, Gorr G (2006) Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns. Appl Microbiol Biotechnol 70:337–345

    Article  PubMed  CAS  Google Scholar 

  • Weise A, Altmann F, Rodriguez-Franco M, Sjoberg ER, Baumer W, Launhardt H, Kietzmann M, Gorr G (2007) High-level expression of secreted complex glycosylated recombinant human erythropoietin in the Physcomitrella Delta-fuc-t Delta-xyl-t mutant. Plant Biotechnol J 5:389–401

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Funding of this work by the German Federal Ministry of Education and Research (BMBF Grants 0312624 and 0313852) and the Excellence Initiative of the German Federal and State Governments (EXC 294) is gratefully acknowledged. We thank Anne Katrin Prowse for proofreading of the manuscript.

Author information

Authors and Affiliations

  1. Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104, Freiburg, Germany

    Eva L. Decker & Ralf Reski

  2. BIOSS Centre for Biological Signalling Studies, 79104, Freiburg, Germany

    Ralf Reski

  3. FRIAS Freiburg Institute for Advanced Studies, 79104, Freiburg, Germany

    Ralf Reski

Authors
  1. Eva L. Decker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ralf Reski
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ralf Reski.

Additional information

Communicated by M. Horn.

A contribution to the Special Issue: Plant Molecular Pharming in 2012 and Beyond

Rights and permissions

Reprints and permissions

About this article

Cite this article

Decker, E.L., Reski, R. Glycoprotein production in moss bioreactors. Plant Cell Rep 31, 453–460 (2012). https://doi.org/10.1007/s00299-011-1152-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1152-5

Keywords

Search

Navigation