Skip to main content
SpringerLink
Log in

Transgenic Expression of Therapeutic Proteins in Arabidopsis thaliana Seed

  • Protocol
  • First Online:
Therapeutic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 899))

Abstract

The production of therapeutic proteins in plant seed augments alternative production platforms such as microbial fermentation, cell-based systems, transgenic animals, and other recombinant plant production systems to meet increasing demands for the existing biologics, drugs under evaluation, and undiscovered therapeutics in the future. We have developed upstream purification technologies for oilseeds which are designed to cost-effectively purify therapeutic proteins amenable to conventional downstream manufacture. A very useful tool in these endeavors is the plant model system Arabidopsis thaliana. The current chapter describes the rationale and methods used to over-express potential therapeutic products in A. thaliana seed for evaluation and definitive insight into whether our production platform, Safflower, can be utilized for large-scale manufacture.

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 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
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. Obembe OO et al (2010) Advances in plant molecular farming. Biotechnol Adv 29(2):210–222

    Article  PubMed  Google Scholar 

  2. Sourrouille C et al (2009) From Neanderthal to nanobiotech: from plant potions to pharming with plant factories. Methods Mol Biol 483:1–23

    Article  PubMed  CAS  Google Scholar 

  3. Ma JK et al (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    Article  PubMed  CAS  Google Scholar 

  4. Twyman RM et al (2003) Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21(12):570–578

    Article  PubMed  CAS  Google Scholar 

  5. Davies HM (2010) Commercialization of whole-plant systems for biomanufacturing of protein products: evolution and prospects. Plant Biotechnol J 8:854–861

    Google Scholar 

  6. Dunwell JM (2011) Foresight project on global food and farming futures. Crop biotechnology: prospects and opportunities. J Agric Sci 149:17–27

    Google Scholar 

  7. Castilho A et al (2010) In planta protein sialylation through overexpression of the respective mammalian pathway. J Biol Chem 285(21):15923–15930

    Article  PubMed  CAS  Google Scholar 

  8. De Muynck B et al (2010) Production of antibodies in plants: status after twenty years. Plant Biotechnol J 8:529–563

    Article  PubMed  Google Scholar 

  9. Loos A et al (2010) Production of monoclonal antibodies with a controlled N-glycosylation pattern in seeds of Arabidopsis thaliana. Plant Biotechnol J 9(2):179–192

    Article  Google Scholar 

  10. Loos A et al (2011) Expression of antibody fragments with a controlled N-glycosylation pattern and induction of ER-derived vesicles in seeds in Arabidopsis thaliana. Plant Physiol 155:2036–2048

    Article  PubMed  CAS  Google Scholar 

  11. Boothe J et al (2010) Seed-based expression systems for plant molecular farming. Plant Biotechnol J 8:588–606

    Article  PubMed  CAS  Google Scholar 

  12. Markley N et al (2006) Producing proteins using transgenic oilbody oleosin technology. Biopharm Int 19:34–47

    CAS  Google Scholar 

  13. The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408:796–815

    Article  Google Scholar 

  14. Koornneef M, Meinke D (2010) The development of Arabidopsis as a model plant. Plant J 61:909–921

    Article  PubMed  CAS  Google Scholar 

  15. Rounsley SD, Last RL (2010) Shotguns and SNPs: how fast and cheap sequencing is revolutionizing plant biology. Plant J 61:922–927

    Article  PubMed  CAS  Google Scholar 

  16. Liu Y-G et al (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8(3):457–463

    Article  PubMed  CAS  Google Scholar 

  17. Nykiforuk CL et al (2006) Transgenic expression and recovery of biologically active recombinant human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 4:77–85

    Article  PubMed  CAS  Google Scholar 

  18. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6):735–743

    Article  PubMed  CAS  Google Scholar 

  19. Van Rooijen GJH, Moloney MM (1995) Structural requirements of oleosin domains for subcellular targeting to the oil body. Plant Physiol 109:1353–1361

    Article  PubMed  Google Scholar 

  20. Hajdukiewicz P et al (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol 25:989–994

    Article  PubMed  CAS  Google Scholar 

  21. Hood EH et al (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168(3):1291–1301

    PubMed  CAS  Google Scholar 

  22. Hellens R et al (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5(10):446–451

    Article  PubMed  CAS  Google Scholar 

  23. Inoue H et al (1990) High efficiency transformation of Escherichia coli with plasmids. Gene 96:23–28

    Article  PubMed  CAS  Google Scholar 

  24. Schneitz K et al (1995) Wild-type ovule development in Arabidopsis thaliana: a light microscope study of cleared whole-mount tissue. Plant J 7(5):731–749

    Article  Google Scholar 

  25. Lee L-Y, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol 146:325–332

    Article  PubMed  CAS  Google Scholar 

  26. Luo ZQ, Farrand SK (1999) Cloning and characterization of a tetracycline resistance determinant present in Agrobacterium tumefaciens C58. J Bacteriol 181:618–626

    PubMed  CAS  Google Scholar 

  27. Cheng AM et al (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17:646–649

    Article  CAS  Google Scholar 

  28. Kjeldsen T et al (2001) Expression of insulin in yeast: the importance of molecular adaptation for secretion and conversion. Biotechnol Genet Eng Rev 18:89–121

    PubMed  CAS  Google Scholar 

  29. Slightom JL et al (1983) Complete nucleotide sequence of a French bean storage protein gene: phaseolin. Proc Natl Acad Sci USA 80:1897–1901

    Article  PubMed  CAS  Google Scholar 

  30. Abell BM et al (2002) Membrane protein topology of oleosin is constrained by its long hydrophobic domain. J Biol Chem 277(10):8602–8610

    Article  PubMed  CAS  Google Scholar 

  31. Abell BM et al (2004) Membrane topology and sequence requirements for oilbody targeting of oleosin. Plant J 37:461–470

    Article  PubMed  CAS  Google Scholar 

  32. Wohlleben W et al (1988) Nucleotide sequence of the phophinothricin N-acetyltransferase gene from Streptomyces viridochromogenes TA1/4494 and its expression in Nicotiana tabacum. Gene 70:25–37

    Article  PubMed  CAS  Google Scholar 

  33. Kawalleck P et al (1993) Polyubiquitin gene expression and structural properties of the ubi4-2 gene in Petroselinum crispum. Plant Mol Biol 21:673–684

    Article  PubMed  CAS  Google Scholar 

  34. Stoger E et al (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotechnol 16:167–173

    Article  PubMed  CAS  Google Scholar 

  35. Rothstein SJ et al (1987) Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation. Gene 53:153–161

    Article  PubMed  CAS  Google Scholar 

  36. Georgiou G, Valax P (1996) Expression of correctly folded proteins in Escherichia coli. Curr Opin Biotechnol 7:190–197

    Article  PubMed  CAS  Google Scholar 

  37. Nykiforuk CL et al (2011) Expression and recovery of biologically active recombinant Apolipoprotein AIMilano from transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol J 9(2):250–263

    Article  PubMed  CAS  Google Scholar 

  38. Kühnel B et al (2003) Precise and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases. Protein Eng 16(10):777–783

    Article  PubMed  Google Scholar 

  39. Schechter I, Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27(2):157–162

    Article  PubMed  CAS  Google Scholar 

  40. Schechter I, Berger A (1968) On the active site of proteases. III. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem Biophys Res Commun 32(5):898–902

    Article  PubMed  CAS  Google Scholar 

  41. Keil B (1992) Specificity of proteolysis. Springer–Verlag, Berlin/Heidelberg/New York, p 335

    Book  Google Scholar 

  42. Nagai K et al (1985) Oxygen binding properties of human mutant hemoglobins synthesized in Escherichia coli. Proc Natl Acad Sci USA 82:7252–7255

    Article  PubMed  CAS  Google Scholar 

  43. LaVallie ER et al (1993) Cloning and functional expression of a cDNA encoding the catalytic subunit of bovine enterokinase. J Biol Chem 268:23311–23317

    PubMed  CAS  Google Scholar 

  44. Geng Y et al (2010) Expression of active recombinant human tissue-type plasminogen activator by using in vivo polyhydroxybutyrate granule display. Appl Environ Microbiol 76(21):7226–7230

    Article  PubMed  CAS  Google Scholar 

  45. Bader O et al (2008) Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans, C. glabrata, Saccharomyces cerevisiae and Pichia pastoris. BMC Microbiol 8:1–16

    Article  Google Scholar 

  46. Nilsson CP et al (1989) Engineering substilisin BPN’ for site-specific proteolysis. Proteins 6(3):240–248

    Article  PubMed  Google Scholar 

  47. Kahle NA et al (2010) Furin cleavage of bacterial expressed glutathione-S-transferase-pro-transforming growth factor β1 fusion protein in vitro. Protein Pept Lett 17(4):416–418

    Article  PubMed  CAS  Google Scholar 

  48. Chong S et al (1997) Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element. Gene 192:277–281

    Article  Google Scholar 

  49. Butt TR et al (2005) SUMO fusion technology for difficult-to-express proteins. Protein Expr Purif 43:1–9

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. SemBioSys Genetics Inc, Calgary, AB, Canada

    Cory L. Nykiforuk & Joseph G. Boothe

Authors
  1. Cory L. Nykiforuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joseph G. Boothe
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cory L. Nykiforuk .

Editor information

Editors and Affiliations

  1. MedImmune, LLC, MedImmune Way One, Gaithersburg, 20878, USA

    Vladimir Voynov

  2. , Department of Physical Biochemistry, Biogen Idec, Cambridge Center 14, Cambridge, 02142, Massachusetts, USA

    Justin A. Caravella

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Nykiforuk, C.L., Boothe, J.G. (2012). Transgenic Expression of Therapeutic Proteins in Arabidopsis thaliana Seed. In: Voynov, V., Caravella, J. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 899. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-921-1_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-921-1_16

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-920-4

  • Online ISBN: 978-1-61779-921-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation