Skip to main content
SpringerLink
Log in

Production of Designer VHH-Based Antibodies in Plants

  • Protocol
  • First Online:
Single-Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2446))

Abstract

Simplified monoclonal antibodies can be produced by fusing a VHH or nanobody, derived from camelid heavy-chain-only antibodies to the Fc domain of either IgG (VHH-IgG), IgA (VHH-IgA), or IgY (VHH-IgY). These recombinant antibodies are encoded by a single gene and their production can be easily scaled up in plants. This chapter contains methods for Gateway cloning of VHH-Fc fusions into the binary T-DNA vectors pEAQ-HT-DEST1 and pPhasGW, electroporation of Agrobacterium with the resulting constructs, transient antibody expression in Nicotiana benthamiana leaves, and stable antibody expression in Arabidopsis thaliana seeds. The properties of chimeric VHH-based antibodies produced in plants enable novel passive immunization treatments, such as in-feed oral delivery or intravenous injection.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Ma JKC, Chikwamba R, Sparrow P et al (2005) Plant-derived pharmaceuticals—the road forward. Trends Plant Sci 10:580–585

    Article  CAS  Google Scholar 

  2. De Greve H, Virdi V, Bakshi S et al (2020) Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization. Curr Opin Biotechnol 61:96–101

    Article  Google Scholar 

  3. Redkiewicz P, Sirko A, Kamel KA et al (2014) Plant expression systems for production of hemagglutinin as a vaccine against influenza virus. Acta Biochim Pol 61:551–560

    Article  Google Scholar 

  4. Kapila J, De Rycke R, Van Montagu M et al (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108

    Article  CAS  Google Scholar 

  5. Dai S, Zheng P, Marmey P et al (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breeding 7:25–33

    Article  CAS  Google Scholar 

  6. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the gene-jockeying tool. Microbiol Mol Biol Rev 67:16–37

    Google Scholar 

  7. Ko K, Koprowski H (2005) Plant biopharming of monoclonal antibodies. Virus Res 111:93–100

    Article  CAS  Google Scholar 

  8. Sainsbury F, Thuenemann EC, Lomonossoff GP (2009) pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7:682–693

    Article  CAS  Google Scholar 

  9. De Jaeger G, Scheffer S, Jacobs A et al (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences. Nat Biotechnol 20:1265–1268

    Article  Google Scholar 

  10. Khan HA, Mutus B (2014) Protein disulfide isomerase a multifunctional protein with multiple physiological roles. Front Chem 2:70

    Google Scholar 

  11. Loos A, Van Droogenbroeck B, Hillmer S et al (2011) Expression of antibody fragments with a controlled N-glycosylation pattern and induction of endoplasmic reticulum-derived vesicles in seeds of Arabidopsis. Cell Biol Sign Trans 155:2036–2048

    CAS  Google Scholar 

  12. Van Droogenbroeck B, De Wilde K, Depicker A (2009) Production of antibody fragments in Arabidopsis seeds. Methods Mol Biol 483:89–101

    Article  Google Scholar 

  13. Virdi V, Coddens A, De Buck S et al (2013) Orally fed seeds producing designer IgAs protect weaned piglets against enterotoxigenic Escherichia coli infection. Proc Natl Acad Sci U S A 110:11809–11814

    Article  CAS  Google Scholar 

  14. Van Droogenbroeck B, Cao J, Stadlmann J et al (2007) Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. Proc Natl Acad Sci U S A 104:1430–1435

    Article  Google Scholar 

  15. Boothe J, Nykiforuk C, Shen Y et al (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8:588–606

    Article  CAS  Google Scholar 

  16. Fiedler U, Conrad U (1995) High-level production and long-term storage of engineered antibodies in transgenic tobacco seeds. Nat Biotechnol 13:1090–1093

    Article  CAS  Google Scholar 

  17. Kawakatsu T, Takaiwa F (2010) Cereal seed storage protein synthesis: fundamental processes for recombinant protein production in cereal grains. Plant Biotechnol J 8:939–953

    Article  CAS  Google Scholar 

  18. Whaley KJ, Hiatt A, Zeitlin L (2011) Emerging antibody products and Nicotiana manufacturing. Hum Vaccin 7:349–356

    Article  CAS  Google Scholar 

  19. Paul M, Ma JKC (2011) Biotechnology and plant-made pharmaceuticals: leading products and production platforms. Biotechnol Appl Biochem 58:58–67

    Article  CAS  Google Scholar 

  20. Peyret H, Lomonossoff GP (2013) The pEAQ vector series: the easy and quick way to produce recombinant proteins in plants. Plant Mol Biol 83:51–58

    Article  CAS  Google Scholar 

  21. Liu L, Grainger J, Cainzares MC et al (2004) Cowpea mosaic virus RNA-1 acts as an amplicon whose effects can be counteracted by a RNA-2-encoded suppressor of silencing. Virology 323:37–48

    Article  CAS  Google Scholar 

  22. Scholthof HB (2006) The Tombusvirus-encoded P19: from irrelevance to elegance. Nat Rev Microbiol 4:405–411

    Article  CAS  Google Scholar 

  23. Saxena P, Hsieh YC, Alvarado VY et al (2011) Improved foreign gene expression in plants using a virus-encoded suppressor of RNA silencing modified to be developmentally harmless. Plant Biotechnol J 9:703–712

    Article  CAS  Google Scholar 

  24. Hoekema A, Hirsch PR, Hooykaas PJJ et al (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    Article  CAS  Google Scholar 

  25. Hellens RP, Edwards EA, Leyland NR et al (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832

    Article  CAS  Google Scholar 

  26. Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396

    Article  CAS  Google Scholar 

  27. Miller JH (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Long Island, pp 439–443

    Google Scholar 

  28. Bertani G (2004) Lysogeny at mid-twentieth century: P1, P2, and other experimental systems. J Bacteriol 186:595–600

    Article  CAS  Google Scholar 

  29. Sainsbury F, Lomonossoff GP (2008) Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148:1212–1218

    Article  CAS  Google Scholar 

  30. Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  CAS  Google Scholar 

  31. McCormac AC, Wu H, Bao M et al (1998) The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.). Euphytica 99:17–25

    Article  CAS  Google Scholar 

  32. De Buck S, Virdi V, De Meyer T et al (2012) Production of camel-like antibodies in plants. Methods Mol Microbiol 911:305–324

    Google Scholar 

  33. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium -mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  Google Scholar 

  34. Griffiths AJF, Miller JH, Suzuki DT (1993) An introduction to genetic analysis, 5th edn. WH Freeman, New York

    Google Scholar 

  35. De Neve M, De Buck S, Jacobs A et al (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11:15–29

    Article  Google Scholar 

  36. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    Article  CAS  Google Scholar 

  37. Vanmarsenille C, Elseviers J, Yvanoff C et al (2018) In planta expression of nanobody-based designer chicken antibodies targeting Campylobacter. PLoS One 13:e0204222

    Google Scholar 

  38. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  39. Goossens A, Dillen W, De Clercq J et al (1999) The arcelin-5 gene of Phaseolus vulgaris directs high seed-specific expression in transgenic Phaseolus acutifolius and Arabidopsis plants. Plant Physiol 120:1095–1104

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. VIB-VUB Center for Structural Biology, Brussels, Belgium

    Henri De Greve

  2. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Henri De Greve

Authors
  1. Henri De Greve
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Henri De Greve .

Editor information

Editors and Affiliations

  1. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Greg Hussack

  2. Human Health Therapeutics Research Centre, National Research Council Canada, Ottawa, ON, Canada

    Kevin A. Henry

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

De Greve, H. (2022). Production of Designer VHH-Based Antibodies in Plants. In: Hussack, G., Henry, K.A. (eds) Single-Domain Antibodies. Methods in Molecular Biology, vol 2446. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2075-5_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2075-5_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2074-8

  • Online ISBN: 978-1-0716-2075-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation