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Evaluation of somatic embryos of alfalfa for recombinant protein expression

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Abstract

Key message

Somatic embryos of alfalfa can accumulate higher levels of recombinant proteins comparing to vegetative organs. Somatic embryos may be explored as a new system for new protein production for plants.

Abstract

Plants have been explored via genetic engineering as an inexpensive system for recombinant protein production. However, protein expression levels in vegetative tissues have been low, which limits the commercial utilization of plant expression systems. Somatic embryos resemble zygotic embryos in many aspects and may accumulate higher levels of proteins as true seed. In this study, somatic embryo of alfalfa (Medicago sativa L.) was investigated for the expression of recombinant proteins. Three heterologous genes, including the standard scientific reporter uid that codes for β-glucuronidase and two genes of interest: ctb coding for cholera toxin B subunit (CTB), and hIL-13 coding for human interleukin 13, were independently introduced into alfalfa via Agrobacterium-mediated transformation. Somatic embryos were subsequently induced from transgenic plants carrying these genes. Somatic embryos accumulated approximately twofold more recombinant proteins than vegetative organs including roots, stems, and leaves. The recombinant proteins of CTB and hIL-13 accumulated up to 0.15 and 0.18 % of total soluble protein in alfalfa somatic embryos, respectively. The recombinant proteins expressed in somatic embryos also exhibited biological activities. As somatic embryos can be induced in many plant species and their production can be scaled up via different avenues, somatic embryos may be developed as an efficient expression system for recombinant protein production.

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Abbreviations

CTB:

Cholera toxin B

ELISA:

Enzyme-linked immunosorbent assay

GUS:

β-Glucuronidase

hIL-13:

Human interleukin 13

References

  • Arakawa T, Chong DK, Merritt JL, Langridge WH (1997) Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res 6:403–413

    Article  CAS  PubMed  Google Scholar 

  • Arcalis E, Stadlmann J, Rademacher T, Marcel S, Sack M, Altmann F, Stoger E (2013) Plant species and organ influence the structure and subcellular localization of recombinant glycoproteins. Plant Mol Biol 83:105–117

    Article  CAS  PubMed  Google Scholar 

  • Bancroft AJ, McKenzie AN, Grencis RK (1998) A critical role for IL-13 in resistance to intestinal nematode infection. J Immunol 160:3453–3461

    CAS  PubMed  Google Scholar 

  • Bardor M, Loutelier-Bourhis C, Paccalet T, Cosette P, Fitchette AC, Vézina LP, Trépanier S, Dargis M, Lemieux R, Lange C, Faye L, Lerouge P (2003) Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures. Plant Biotechnol J 1:451–462

    Article  CAS  PubMed  Google Scholar 

  • Barta A, Sommergruber K, Thompson D, Hartmuth K, Matzke MA, Matzke AJM (1986) The expression of a nopaline synthase-human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue. Plant Mol Biol 6:347–357

    Article  CAS  PubMed  Google Scholar 

  • Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Blaydes DF (1966) Interaction of kinetin and various inhibitors in the growth of soybean tissues. Physiol Plant 19:748–753

    Article  CAS  Google Scholar 

  • Chanvivattana Y, Bishipp A, Schubert D, Stock C, Moon YH, Sung ZR, Goodrich J (2004) Interaction of Polycomb-group proteins controlling flowering in Arabidopsis. Development 131:5263–5276

    Article  CAS  PubMed  Google Scholar 

  • Conley AJ, Mohib K, Jevnikar AM, Brandle JE (2009) Plant recombinant erythropoietin attenuates inflammatory kidney cell injury. Plant Biotechnol J 7:183–199

    Article  CAS  PubMed  Google Scholar 

  • Copeland LO, McDonald MB (2001) Seed science and technology. Kluwer, Norwell, USA, pp 32–56

    Google Scholar 

  • Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6:219–226

    Article  CAS  PubMed  Google Scholar 

  • Du S, Erickson L, Bowley S (1994) Effect of plant genotype on the transformation of cultivated alfalfa (Medicago sativa) by Agrobacterium tumefaciens. Plant Cell Rep 13:330–334

    Article  CAS  PubMed  Google Scholar 

  • Düring K, Hippe S, Kreuzaler F, Schell J (1990) Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum. Plant Mol Biol 15:281–293

    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  CAS  PubMed  Google Scholar 

  • Hiatt A, Cafferkey R, Bowdish K (1989) Production of antibodies in transgenic plants. Nature 342:76–78

    Article  CAS  PubMed  Google Scholar 

  • Holmgren J, Lycke N, Czerkinsky C (1993) Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems. Vaccine 11:1179–1184

    Article  CAS  PubMed  Google Scholar 

  • Jani D, Meena LS, Rizwan-ul-Haq QM, Singh Y, Sharma AK, Tyagi AK (2002) Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res 11:447–454

    Article  CAS  PubMed  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS-fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kang TJ, Lee WS, Choi EG, Kim JW, Kim BG, Yang MS (2006) Mass production of somatic embryos expressing Escherichia coli heat-labile enterotoxin B subunit in Siberian ginseng. Biotechnol J 121:124–133

    Article  CAS  Google Scholar 

  • Kim YS, Kim BG, Kim TG, Kang TJ, Yang MS (2006) Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformed system. Plant Cell Tissue Organ Cult 87:203–210

    Article  CAS  Google Scholar 

  • Kim YS, Kim MY, Kim TG, Yang MS (2009) Expression and assembly of cholera toxin B subunit (CTB) in transgenic carrot (Daucus carota L.). Mol Biotechnol 41:8–14

    Article  PubMed  Google Scholar 

  • Krochko JE, Bantroch DJ, Greenwood JS, Bewley JD (1994) Seed storage proteins in developing somatic embryos of alfalfa: defects in accumulation compared to zygotic embryos. J Exp Bot 45:699–708

    Article  CAS  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 

  • Li D, O’Leary J, Huang Y, Huner NP, Jevnikar AM, Ma S (2006) Expression of cholera toxin B subunit and the B chain of human insulin as a fusion protein in transgenic tobacco plants. Plant Cell Rep 25:417–424

    Article  PubMed  Google Scholar 

  • Liu W, Liang Z, Sibbald S, Hunter D, Tian L (2013) Preservation and faithful expression of transgene via artificial seeds in alfalfa. PLoS ONE 8(5):e56699. doi:10.1371/journal.pone.0056699

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ma JK, Drake PM, Christou P (2003) The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 4:794–805

    Article  CAS  PubMed  Google Scholar 

  • McKersie BD, Murnagham KS, Jones KS, Bowley SR (2000) Iron-superoxide dismutase expression in transgenic alfalfa increases winter survival without a detectable increase in photosynthetic oxidative stress tolerance. Plant Physiol 122:1427–1437

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Melnik S, Stoger E (2013) Green factories for biopharmaceuticals. Curr Med Chem 20:1038–1046

    CAS  PubMed  Google Scholar 

  • Minty A, Chalon P, Derocq JM, Dumont X, Guillemot JC, Kaghad M, Labit C, Leplatois P, Liauzun P, Miloux B (1993) Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses. Nature 362:248–250

    Article  CAS  PubMed  Google Scholar 

  • Montaner LJ, Doyle AG, Collin M, Herbein G, Illei P, James W, Minty A, Caput D, Ferrara P, Gordon S (1993) Interleukin 13 inhibits human immunodeficiency virus type 1 production in primary blood-derived human macrophages in vitro. J Exp Med 178:743–747

    Article  CAS  PubMed  Google Scholar 

  • Munro S, Pelham HR (1987) A C-terminal signal prevents secretion of luminal ER proteins. Cell 48:899–907

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812

    Article  CAS  PubMed  Google Scholar 

  • Ramessar K, Sabalza M, Capell T, Christou P (2008) Maize plants: an ideal production platform for effective and safe molecular pharming. Plant Sci 174:409–419

    Article  CAS  Google Scholar 

  • Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204

    Article  CAS  Google Scholar 

  • Schubert D, Clarenz O, Goodrich J (2005) Epigenetic control of plant development by polycomb-group proteins. Curr Opin Plant Biol 8:553–561

    Article  CAS  PubMed  Google Scholar 

  • Senaratna T (1992) Artificial seeds. Biotech Adv 20:379–392

    Article  Google Scholar 

  • Senaratna T, McKersie BD, Bowley SR (1990) Artificial seeds of alfalfa (Medicago sativa L.) induction of desiccation tolerance in somatic embryos. In Vitro Cell Dev Biol 26:85–90

    Article  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 

  • Shimamura T, Husain SR, Puri RK (2006) The IL-4 and IL-13 pseudomonas exotoxins: new hope for brain tumor therapy. Neurosurg Focus 20:E11

    Article  PubMed  Google Scholar 

  • Stanya KJ, Jacobi D, Liu S, Bhargava P, Dai L, Gangl MR, Inouye K, Barlow JL, Ji Y, Mizgerd JP, Qi L, Shi H, McKenzie AN, Lee CH (2013) Direct control of hepatic glucose production by interleukin-13 in mice. J Clin Invest 123:261–271

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Streatfield SJ (2007) Approaches to achieve high-level heterologous protein production in plants. Plant Bio J 1:2–15

    Article  Google Scholar 

  • Tesfaye M, Denton MD, Samac DA, Vance CP (2005) Transgenic alfalfa secretes a fungal endochitinase protein to the rhizosphere. Plant Soil 269:233–243

    Article  CAS  Google Scholar 

  • Tian L, Wang H, Wu K, Latoszek-Green M, Hu M, Miki B, Brown D (2002) Efficient recovery of transgenic plants through organogenesis and embryogenesis using a cryptic promoter to drive marker gene expression. Plant Cell Rep 20:1181–1187

    Article  CAS  Google Scholar 

  • Tremblay R, Wang D, Jevnikar AM, Ma S (2010) Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 28:214–221

    Article  CAS  PubMed  Google Scholar 

  • Twyman RM, Schillberg S, Fischer R (2005) Transgenic plants in the biopharmaceutical market. Expert Opin Emerg Drugs 10:185–218

    Article  CAS  PubMed  Google Scholar 

  • Wang XG, Zhang GH, Liu CX, Zhang YH, Xiao CZ, Fang RX (2001) Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity. Biotechnol Bioeng 72:490–494

    Article  CAS  PubMed  Google Scholar 

  • Wang DJ, Brandsma M, Yin Z, Wang A, Jevnikar AM, Ma S (2008) A novel platform for biologically active recombinant human interleukin-13 production. Plant Biotechnol J 6:504–515

    Article  CAS  PubMed  Google Scholar 

  • Webster DE, Thomas MC (2012) Post-translational modification of plant-made foreign proteins; glycosylation and beyond. Biotechnol Adv 30:410–418

    Article  CAS  PubMed  Google Scholar 

  • Wilde HD, Nelson WS, Booij H, Vries SC, Thomas TL (1988) Gene-expression programs in embryogenic and non-embryogenic carrot cultures. Planta 176:205–211

    Article  CAS  PubMed  Google Scholar 

  • Winkelmann T, Heintz D, Van Dorsselaer A, Serek M, Braun HP (2006) Proteomic analyses of somatic and zygotic embryos of Cyclamen persicum Mill. reveal new insights into seed and germination physiology. Planta 224:508–519

    Article  CAS  PubMed  Google Scholar 

  • Ziegelhoffer T, Will J, Austin-Phillips S (1999) Expression of bacterial cellulase genes in transgenic alfalfa (Medicago sativa L.), potato (Solanum tuberosum L.) and tobacco (Nicotiana tabacum L.). Mol Breed 5:309–318

    Article  CAS  Google Scholar 

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Acknowledgments

We thank Dr. R. Menassa for providing pCAMter X vector. We thank S. Sawhney for technical support.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Biology, University of Western Ontario, London, ON, Canada

    Guohua Fu & Vojislava Grbic

  2. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, ON, Canada

    Lining Tian

  3. Platigen Inc., London, ON, Canada

    Shengwu Ma

Authors
  1. Guohua Fu
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  2. Vojislava Grbic
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  3. Shengwu Ma
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  4. Lining Tian
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Corresponding author

Correspondence to Lining Tian.

Additional information

Communicated by Zeng-Yu Wang.

Electronic supplementary material

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299_2014_1700_MOESM1_ESM.docx

Supp. Figure 1. Analysis of alfalfa transformation for uid gene by multiplex PCR. Neomycin phosphotransferase II (npt II) and uid specific primers, generating 700 bp and 198 bp fragments, respectively, were used. Lane1 to 12: PCR products with DNA template from independent transgenic lines. + and -, positive control using pCAMBIA 2301 vector as a PCR template and negative control, respectively. The 100 bp DNA ladder is indicated. (DOCX 176 kb)

299_2014_1700_MOESM2_ESM.docx

Supp. Figure 2. Analysis of alfalfa transformation for ctb by multiplex PCR. Neomycin phosphotransferase II (npt II) and ctb-specific primers, generating 700 bp and 198 bp fragments, respectively, were used. Lane1 to 22: PCR products with DNA template from independent lines; +  and -: positive control using pBI-CTB vector as a PCR template and negative control, respectively. The 100 bp DNA ladder is indicated. In total, 38 out of 41 plants carried the ctb gene and the figure shows part of the PCR analysis results. (DOCX 176 kb)

299_2014_1700_MOESM3_ESM.docx

Supp. Figure 3. Analysis of alfalfa transformation for hIL-13 by multiplex PCR. Neomycin phosphotransferase II (npt II) and hIL-13 specific primers, generating 700 bp and 415 bp fragments respectively, were used. Lane 1 to 15: independent lines. + and -, positive control using pCAMBIA hIL 13-GFP vector as a PCR template and negative control, respectively. The 100 bp DNA ladder is indicated. (DOCX 109 kb)

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Fu, G., Grbic, V., Ma, S. et al. Evaluation of somatic embryos of alfalfa for recombinant protein expression. Plant Cell Rep 34, 211–221 (2015). https://doi.org/10.1007/s00299-014-1700-x

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  • DOI: https://doi.org/10.1007/s00299-014-1700-x

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