Skip to main content
SpringerLink
Log in

Deposition mode of transforming growth factor-β expressed in transgenic rice seed

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Mouse TGF-β highly accumulated by expressing as a secretory homodimeric protein in transgenic rice endosperm. It was tightly deposited in ER-derived PBs by interaction with cysteine-rich prolamins.

Abstract

TGF-β is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the induction of regulatory T cells. In order to utilize rice-based TGF-β as a tool to promote oral immune tolerance induction, high production of TGF-β is essentially required. When the codon-optimized mTGF-β was expressed as a secretory protein by ligating an N-terminal signal peptide and C-terminal KDEL ER retention signal under the control of the endosperm-specific rice storage protein glutelin GluB-1 promoter, accumulation level was low in stable transgenic rice seeds. Then, to increase the accumulation level of mTGF-β, it was expressed as fusion proteins by inserting into the C terminus of acidic subunit of glutelin GluA and the variable region of 26 kDa globulin. When fused with the glutelin, it could accumulate well as visible bands by CBB staining gel, but not for the 26 kDa globulin. Unexpectedly, expression of homodimeric mTGF-β linked by a 6×Gly1×Ser linker as secretory protein resulted in higher level of accumulation. This expression level was further enhanced by reduction of some endogenous prolamins by RNA interference. The monomeric and dimeric mTGF-βs were deposited in ER-derived PBs containing prolamins. When highly produced in rice seed, it is notable that most of ER-derived PBs were distorted and granulated. Step-wise extraction of storage proteins from rice seeds suggested that the mTGF-β strongly interacted with cysteine-rich prolamins via disulfide bonds. This result was also supported by the finding that reducing agent was absolutely required for mTGF-β extraction.

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
Fig. 8

Similar content being viewed by others

Abbreviations

BiP:

Binding protein

CHO cell:

Chinese hamster ovary cell

CTAB:

Cetyl trimethyl ammonium bromide

Cys-rich prolamins:

Cysteine-rich prolamins

DTT:

Dithiothreitol

ER:

Endoplasmic reticulum

GALT:

Gut-associated lymphoid tissue

GFP:

Green fluorescent protein

PB:

Protein body

2-MER:

2-Mercaptoethanol

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

TGF-β:

Transforming growth factor

References

  • Cabanos C, Kato N, Amari Y, Fujiwara K, Ohno T, Shimizu K, Goto T, Shimada M, Kuroda M, Masuda T, Takaiwa F, Utsumi S, Nagaoka S, Maruyama N (2012) Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α’ subunit of soybean β-conglycinin. Transgenic Res 23:609–620

    Article  CAS  Google Scholar 

  • Conley AJ, Joensuu JJ, Richman A, Menassa R (2011) Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants. Plant Biteochnol J 9:419–433

    Article  CAS  Google Scholar 

  • Da Cunha NB, Vianna GR, Lima TA, Rech E (2014) Molecular farming of human cytokines and blood products from plants: challenges in biosynthesis and detection of plant-produced recombinant proteins. Biotechnol J 9:39–50

    Article  CAS  PubMed  Google Scholar 

  • Derynck R, Jarrett J, Chen E, Eaton D, Bell J, Assoian R, Roberts A, Sporn M, Goeddel D (1985) Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature 316:701–715

    Article  CAS  PubMed  Google Scholar 

  • Desai PN, Shrivastava N, Padh H (2010) Production of heterologous proteins in plants: strategies for optimal expression. Biotechnol Adv 28:427–435

    Article  CAS  PubMed  Google Scholar 

  • Fujiwara Y, Yang L, Takaiwa F, Sekikawa K (2016) Expression and purification of recombinant mouse interleukin-4 and -6 from transgenic rice seeds. Mol Biotechnol 58:223–231

    Article  CAS  PubMed  Google Scholar 

  • Goto F, Yoshihara T, Sugimoto N, Toki S, Takaiwa F (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17:282–286

    Article  CAS  PubMed  Google Scholar 

  • Hofbauer A, Stoger E (2013) Subellular accumulation and modification of pharmaceutical proteins in different plant tissues. Curr Pharm Des 19:5495–5502

    Article  CAS  PubMed  Google Scholar 

  • Kawakatsu T, Wakasa Y, Yasuda H, Takaiwa F (2012) RNA silencing induced by an artificial sequence that prevents proper transcription termination in rice. Plant Physiol 160:601–612

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khan I, Twyman RM, Arcalis E, Stoger E (2012) Using storage organelles for the accumulation and encapsulation of recombinant proteins. Biotechnol J 7:1099–1108

    Article  CAS  PubMed  Google Scholar 

  • Kingsley DM (1994) The TGF-β superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev 8:133–146

    Article  CAS  PubMed  Google Scholar 

  • Lau OS, Sun SSM (2009) Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv 27:1015–1022

    Article  CAS  PubMed  Google Scholar 

  • Mucida D, Park Y, Cheroutre H (2009) From the diet to the nucleus: vitamin A and TGF-β join efforts at the mucosal interface of the intestine. Semin Immunol 21:14–21

    Article  CAS  PubMed  Google Scholar 

  • Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, Nakanishi U, Matsumura A, Uozumi A, Hiroi T, Morita S, Tanaka K, Takaiwa F, Kiyono H (2007) Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Acad Sci USA 104:10986–10991

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Onda Y, Nagamine A, Sakurai M, Kumamaru T, Ogawa M, Kawagoe Y (2011) Distinct roles of protein disulfide isomerase and P5 sulfhydryl oxidoreductases in multiple pathways for oxidation of structurally diverse storage proteins in rice. Plant Cell 23:210–223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Oono Y, Wakasa Y, Hirose S, Yang L, Sakuta C, Takaiwa F (2010) Analysis of ER stress in developing rice endosperm accumulating β-amyloid peptide. Plant Biotechnol J 8:691–718

    Article  CAS  PubMed  Google Scholar 

  • Seigneurin-Berny D, Rolland N, Garin J, Joyard J (1999) Differential extraction of hydrophobic proteins from chloroplast envelope membranes: a subcellular-specific proteomic approach to identify rare intrinsic membrane proteins. Plant J 19:217–228

    Article  CAS  PubMed  Google Scholar 

  • Sharma AK, Sharma MK (2009) Plants as bioreactors: recent developments and emerging opportunities. Biotechnol Adv 27:811–832

    Article  CAS  PubMed  Google Scholar 

  • Sirko A, Vanek T, Gora-Sochacka A, Redkiewicz P (2011) Recombinant cytokines from plants. Int J Mol Sci 12:3536–3552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stoger E, Ma JK, Fischer R, Christou P (2005) Sowing the seeds of success: pharmaceutical proteins from plants. Curr Opin Biotechnol 16(2):167–173

    Article  CAS  PubMed  Google Scholar 

  • Streatfield SJ (2007) Appraches to achieve high-level heterologous protein production in plants. Plant Biotechnol J 5:2–15

    Article  CAS  PubMed  Google Scholar 

  • Sugita K, Endo-Kasahara S, Tada Y, Lijun Y, Yasuda H, Hayashi Y, Jomori T, Ebinuma H, Takaiwa F (2005) Genetically modified rice seeds accumulating GLP-1 analogue stimulate insulin secretion from a mouse pancreatic beta-cell line. FEBS Lett 579:1085–1088

    Article  CAS  PubMed  Google Scholar 

  • Tada Y, Usumi S, Takaiwa F (2003) Foreign gene products can be enhanced by introduction into storage protein mutants. Plant Biotechnol J 1:411–422

    Article  CAS  PubMed  Google Scholar 

  • Takagi H, Saito S, Yang L, Nagasaka S, Nishizawa N, Takaiwa F (2005) Oral immunotherapy against a pollen allergy using a seed-based peptide vaccine. Plant Biotechnol J 3:521–533

    Article  CAS  PubMed  Google Scholar 

  • Takagi H, Hiroi T, Hirose S, Yang L, Takaiwa F (2010) Rice seed ER-derived protein body as an efficient delivery vehicle for oral tolerogenic peptides. Peptides 31:1421–1425

    Article  CAS  PubMed  Google Scholar 

  • Takagi H, Watanabe N, Hiroi T, Takaiwa F (2015) Efficacy of transgenic rice containing human interleukin-10 in experimental mouse models of colitis and pollen allergy. Plant Biotechnol 32(4):329–332

    Article  CAS  Google Scholar 

  • Takaiwa F (2013a) Update on the use of transgenic rice seeds in oral immunotherapy. Immunotherapy 5:301–312

    Article  CAS  PubMed  Google Scholar 

  • Takaiwa F (2013b) Increasing the production yield of recombinant protein in transgenic seeds by expanding the deposition space within the intracellular compartment. Bioengineered 4:136–139

    Article  PubMed  PubMed Central  Google Scholar 

  • Takaiwa F, Yang L, Yasuda H (2008) Health-promoting transgenic rice seeds as a direct delivery system for bioactive peptides in human health. In: Hirano HY, Hirai A, Sano Y, Sasaki T (eds) Rice biology in the genomics era. Springer, Berlin, pp 357–373

    Chapter  Google Scholar 

  • Takaiwa F, Hirose S, Takagi H, Yang L, Wakasa Y (2009) Deposition of a recombinant peptide in ER-derived protein bodies by retention with cysteine-rich prolamins in transgenic rice seed. Planta 229:1147–1158

    Article  CAS  PubMed  Google Scholar 

  • Wakasa Y, Takaiwa F (2013) The use of rice seeds to produce human pharmaceuticals for oral therapy. Biotechnol J 8:1133–1143

    Article  CAS  PubMed  Google Scholar 

  • Wakasa Y, Yasuda H, Takaiwa F (2006) High accumulation of bioactive peptide in transgenic rice seeds by expression of introduced multiple genes. Plant Biotechnol J 4:499–510

    CAS  PubMed  Google Scholar 

  • Wakasa Y, Yasuda H, Oono Y, Kawakatsu T, Hirose S, Takahashi H, Hayashi S, Yang L, Takaiwa F (2011) Expression of ER quality control related genes in response to changes in BiP1 levels in developing rice endosperm. Plant J 65:675–689

    Article  CAS  PubMed  Google Scholar 

  • Wang S, Takahashi H, Kajiura H, Kawakatsu T, Fujiyama K, Takaiwa F (2013) Transgenic rice seeds accumulating recombinant hypoallergenic birch pollen allergen Bet v 1 generate giant protein bodies. Plant Cell Physiol 54:917–933

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Suzuki K, Hirose S, Wakasa Y, Takaiwa F (2007) Development of transgenic rice seed accumulating a major Japanese cedar pollen allergen (Cry j 1) structurally disrupted for oral immunotherapy. Plant Biotechnol J 5:815–826

    Article  CAS  PubMed  Google Scholar 

  • Yang L, Hirose S, Takahashi H, Kawakatsu T, Takaiwa F (2012) Recombinant protein yield in rice seed is enhanced by specific suppression of endogenous seed proteins at the same deposit site. Plant Biotechnol J 10:1035–1045

    Article  CAS  PubMed  Google Scholar 

  • Yasuda H, Hirose S, Kawakatsu T, Wakasa Y, Takiwa F (2009) Overexpression of BiP has inhibitory effects on the accumulation of seed storage proteins in endosperm cells of rice. Plant Cell Physiol 50:1532–1543

    Article  CAS  PubMed  Google Scholar 

  • Yoshimura A, Wakabayashi Y, Mori T (2010) Cellular and molecular basis for the regulation of inflammation by TGF-beta. J Biochem 147:781–792

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank Ms. M. Utsuno, Y. Ikemoto, K. Miyashita, and Y. Yajima for technical assistance. This work was supported by ‘Genomics and Agricultural Innovation, GMC0004’ to F.T. from the Ministry of Agriculture Forestry and Fisheries of Japan.

Author information

Authors and Affiliations

  1. Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602, Japan

    Fumio Takaiwa, Lijun Yang, Yuhya Wakasa & Kenjiro Ozawa

  2. Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto, 611-0011, Japan

    Nobuyuki Maruyama

Authors
  1. Fumio Takaiwa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lijun Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nobuyuki Maruyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yuhya Wakasa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kenjiro Ozawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fumio Takaiwa.

Ethics declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Additional information

Communicated by S. Schillberg.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 366 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Takaiwa, F., Yang, L., Maruyama, N. et al. Deposition mode of transforming growth factor-β expressed in transgenic rice seed. Plant Cell Rep 35, 2461–2473 (2016). https://doi.org/10.1007/s00299-016-2047-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-2047-2

Keywords

Search

Navigation