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Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2

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Abstract

The bone morphogenetic protein BMP2 plays a crucial role in the formation and regeneration of bone and cartilage, which is critical for maintaining skeletal integrity and bone fracture repair. Because of its important role in osteogenic properties it has been commercially produced for clinical use. Here we report attempts to express human BMP2 using two plant systems (lettuce chloroplast and soybean seeds). The rhBMP2 gene (coding for the 13 kDa active polypeptide) was introduced in two regions of the lettuce chloroplast genome. Two homoplasmic events were achieved and RT-PCR demonstrated that the BMP2 gene was transcribed. However, it was not possible to detect accumulation of rhBMP2 in leaves. Two soybean events were achieved to express a full-length hBMP2 gene (coding for the 45 kDa pro-BMP2) fused with the α-coixin signal peptide, under control of the β-conglycinin promoter. Pro-BMP2 was expressed in the transgenic seeds at levels of up to 9.28% of the total soluble seed protein as determined by ELISA. It was demonstrated that this recombinant form was biologically active upon administration to C2C12 cell cultures, because it was able to induce an osteogenic cascade, as observed by the enhanced expression of SP7 (osterix) and ALPI (alkaline phosphatase) genes. Collectively, these results corroborated our previous observation that soybean seeds provide an effective strategy for achieving stable accumulation of functional therapeutic proteins, such as BMP2.

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References

  • Ahmad N, Michoux F, Lössl AG, Nixon PJ (2016) Challenges and perspectives in commercializing plastid transformation technology. J Exp Bot 67:5945–5960

    Article  CAS  PubMed  Google Scholar 

  • Apel W, Schulze WX, Bock R (2010) Identification of protein stability determinants in chloroplasts. Plant J 63:636–650

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Aragão FJL, Sarokin L, Vianna GR, Rech EL (2000) Selection of transgenic meristematic cells utilizing a herbicidal molecule results in the recovery of fertile transgenic soybean [Glycine max (L.) Merril] plants at a high frequency. Theor Appl Genet 101:1–6

    Article  Google Scholar 

  • Arlen PA, Falconer R, Cherukumilli S et al (2007) Field production and functional evaluation of chloroplast-derived interferon-α2b. Plant Biotechnol J 5:511–525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bessho K, Konishi Y, Kaihara S, Fujimura K, Okubo Y, Iizuka T (2000) Bone induction by Escherichia coli-derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2. Br J Oral Maxillofac Surg 38:645–649

    Article  CAS  PubMed  Google Scholar 

  • Bock R (2014) Genetic engineering of the chloroplast: novel tools and new applications. Curr Opin Biotechnol 26:7–13

    Article  CAS  PubMed  Google Scholar 

  • 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  PubMed  Google Scholar 

  • Boyhan D, Daniell H (2011) Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide. Plant Biotechnol J 9:585–598

    Article  CAS  PubMed  Google Scholar 

  • Brazil DP, Church RH, Surae S, Godson C, Martin F (2015) BMP signalling: agony and antagony in the family. Trends Cell Biol 25:249–264

    Article  CAS  Google Scholar 

  • Carreira AC, Lojudice FH, Halcsik E, Navarro RD, Sogayar MC, Granjeiro JM (2014a) Bone morphogenetic proteins: facts, challenges, and future perspectives. J Dent Res 93:335–345

    Article  CAS  PubMed  Google Scholar 

  • Carreira AC, Alves GG, Zambuzzi WF, Sogayar MC, Granjeiro JM (2014b) Bone morphogenetic proteins: structure, biological function and therapeutic applications. Arch Biochem Biophys 561:64–73

    Article  CAS  Google Scholar 

  • Ceresoli V, Mainieri D, Del Fabbro M, Weinstein R, Pedrazzini E (2016) A fusion between domains of the human bone morphogenetic protein-2 and maize 27 kD γ-zein accumulates to high levels in the endoplasmic reticulum without forming protein bodies in transgenic tobacco. Front Plant Sci 7:1–13

    Article  Google Scholar 

  • Cunha NB, Araújo ACG, Leite A, Murad AM, Vianna GR, Rech EL (2010) Correct targeting of proinsulin in protein storage vacuoles of transgenic soybean seeds. Genet Mol Res 9:1163–1170

    Article  CAS  PubMed  Google Scholar 

  • Cunha NB, Murad AM, Cipriano TM et al (2011a) Expression of functional recombinant human growth hormone in transgenic soybean seeds. Transgenic Res 20(4):811–826

    Article  CAS  PubMed  Google Scholar 

  • Cunha NB, Murad AM, Ramos GL et al (2011b) Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds. Transgenic Res 20:841–855

    Article  CAS  PubMed  Google Scholar 

  • Daniell H, Singh ND, Mason H, Streatfield SJ (2010) Plant-made vaccine antigens and biopharmaceuticals. Trends Plant Sci 14:669–679

    Article  CAS  Google Scholar 

  • Davoodi-Semiromi A, Schreiber M, Nalapalli S et al (2010) Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery. Plant Biotechnol J 8:223–242

    Article  CAS  PubMed  Google Scholar 

  • De Marchis F, Pompa A, Bellucci M (2012) Plastid proteostasis and heterologous protein accumulation in transplastomic plants. Plant Physiol 160:571–581

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Martinis D, Rybicki EP, Fujiyama K, Franconi R, Benvenuto E (2016) Plant molecular farming: fast, scalable, cheap, sustainable. Front Plant Sci 7:1148

    Article  PubMed  PubMed Central  Google Scholar 

  • Elghabi Z, Karcher D, Zhou F, Ruf S, Bock R (2011) Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome. Plant Biotechnol J 9:599–608

    Article  CAS  PubMed  Google Scholar 

  • Farran I, Río-Manterola F, Íñiguez M, Gárate S, Prieto J, Mingo-Castel AM (2008) High-density seedling expression system for the production of bioactive human cardiotrophin-1, a potential therapeutic cytokine, in transgenic tobacco chloroplasts. Plant Biotechnol J 6:516–527

    Article  CAS  PubMed  Google Scholar 

  • Fernández-San Millán A, Ortigosa SM, Hervás-Stubbs S, Corral-Martínez P, Seguí-Simarro JM, Gaétan J, Coursaget P, Veramendi J (2008) Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol J 6:427–441. https://doi.org/10.1111/j.1467-7652.2008.00338.x

    Article  CAS  PubMed  Google Scholar 

  • Fu T-S, Chang Y-H, Wong C-B et al (2015) Mesenchymal stem cells expressing baculovirus-engineered BMP-2 and VEGF enhance posterolateral spine fusion in a rabbit model. Spine J 15:2036–2044

    Article  PubMed  Google Scholar 

  • Gisby MF, Mellors P, Madesis P et al (2011) A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule. Plant Biotechnol J 9:618–628

    Article  CAS  PubMed  Google Scholar 

  • Glenz K, Bouchon B, Stehle T, Wallich R, Simon MM, Warzecha H (2006) Production of a recombinant bacterial lipoprotein in higher plant chloroplasts. Nat Biotechnol 24:76–77

    Article  CAS  PubMed  Google Scholar 

  • Jin S, Daniell H (2015) The engineered chloroplast genome just got smarter. Trends Plant Sci 20:622–640

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kanamoto H, Yamashita A, Asao H et al (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15:205–217

    Article  CAS  PubMed  Google Scholar 

  • Krichevsky A, Meyers B, Vainstein A, Maliga P, Citovsky V (2010) Autoluminescent plants. PLoS ONE 5:1–6

    Article  CAS  Google Scholar 

  • Lacorte C, Vianna G, Aragão FJL, Rech EL (2010) Molecular characterization of genetically manipulated plants. Plant Cell Cult Essent Methods. https://doi.org/10.1002/9780470686522.ch14

    Article  Google Scholar 

  • Lee M-H, Kwon T-G, Park H-S, Wozney JM, Ryoo H-M (2003) BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun 309:689–694

    Article  CAS  Google Scholar 

  • Lee HL, Park HJ, Kwon A et al (2014) Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation. Exp Cell Res 323:276–287

    Article  CAS  PubMed  Google Scholar 

  • Leelavathi S, Reddy VS (2003) Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors. Mol Breed 11:49–58

    Article  CAS  Google Scholar 

  • Lelivelt CL, McCabe MS, Newell CA, Desnoo CB, van Dun KM, Birch-Machin I, Gray JC, Mills KH, Nugent JM (2005) Stable plastid transformation in lettuce (Lactuca sativa L.). Plant Mol Biol 58:763–774

    Article  CAS  PubMed  Google Scholar 

  • Lim S, Ashida H, Watanabe R et al (2011) Production of biologically active human thioredoxin 1 protein in lettuce chloroplasts. Plant Mol Biol 76:335–344

    Article  CAS  PubMed  Google Scholar 

  • Lissenberg-Thunnissen SN, De Gorter DJJ, Sier CFM, Schipper IB (2011) Use and efficacy of bone morphogenetic proteins in fracture healing. Int Orthop 35:1271–1280

    Article  PubMed  PubMed Central  Google Scholar 

  • Livak KJ (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCt method. Methods 25:402–408

    Article  CAS  Google Scholar 

  • Lomonossoff GP, D’Aoust MA (2016) Plant-produced biopharmaceuticals: a case of technical developments driving clinical deployment. Science 353:1237–1240

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Maldaner FR, Aragão FJL, Dos Santos FB et al (2013) Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis. Appl Microbiol Biotechnol 97:5721–5729

    Article  CAS  PubMed  Google Scholar 

  • Maruoka Y, Oida S, Iimura T, Takeda K, Asahina I, Enomoto S, Sasaki S (1995) Production of functional human bone morphogenetic protein-2 using a baculovirus/Sf-9 insect cell system. Biochem Mol Biol Int 35:957–963

    CAS  PubMed  Google Scholar 

  • Mendonça G, Baccelli D, Mendonça S (2013) Effect of hydrophilic implant surfaces on differentiation of human mesenchymal stem cells. Implant News Perio 10:97–102

    Google Scholar 

  • Nadai M, Bally J, Vitel M et al (2009) High-level expression of active human alpha1-antitrypsin in transgenic tobacco chloroplasts. Transgenic Res 18:173–183

    Article  CAS  PubMed  Google Scholar 

  • Oey M, Lohse M, Kreikemeyer B, Bock R (2009) Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic. Plant J 57:436–445

    Article  CAS  PubMed  Google Scholar 

  • Rech EL, Vianna GR, Aragão FJL (2008) High-efficiency transformation by biolistics of soybean, common bean and cotton transgenic plants. Nat Protoc 3:410–418

    Article  CAS  PubMed  Google Scholar 

  • Sondag GR, Salihoglu S, Lababidi SL et al (2014) Osteoactivin induces transdifferentiation of C2C12 myoblasts into osteoblasts. J Cell Physiol 229:955–966

    Article  CAS  PubMed  Google Scholar 

  • Staub JM, Garcia B, Graves J et al (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Su J, Zhu L, Sherman A et al (2015) Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B. Biomaterials 70:84–93

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Suo G, Chen B, Zhang J, Gao Y, Wang X, He Z, Dai J (2006) Expression of active hBMP2 in transgenic tobacco plants. Plant Cell Rep 25:1316–1324

    Article  CAS  PubMed  Google Scholar 

  • Tabar MS, Habashi AA, Memari HR (2013) Human granulocyte colony-stimulating factor (hg-csf) expression in plastids of Lactuca sativa. Iran Biomed J 17:158–164

    CAS  Google Scholar 

  • Tsuji K, Bandyopadhyay A, Harfe BD, Cox K, Kakar S, Gerstenfeld L, Rosen V (2006) BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing. Nat Genet 38:1424–1429

    Article  CAS  PubMed  Google Scholar 

  • Urist MR (1965) Bone: formation by autoinduction. Science 150:893–899

    Article  CAS  PubMed  Google Scholar 

  • Verma D, Moghimi B, LoDuca PA et al (2010) Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice. Proc Natl Acad Sci 107:7101–7106

    Article  PubMed  Google Scholar 

  • Vianna GR, Cunha NB, Murad AM, Rech EL (2011) Soybeans as bioreactors for biopharmaceuticals and industrial proteins. Genet Mol Res 10:1733–1752

    Article  CAS  PubMed  Google Scholar 

  • Von Einem S, Erler S, Bigl K, Frerich B, Schwarz E (2011) The pro-form of BMP-2 exhibits a delayed and reduced activity when compared to mature BMP-2. Growth Factors 29:63–71

    Article  CAS  Google Scholar 

  • Walsh G (2014) Biopharmaceutical benchmarks. Nat Biotechnol 24:831–833

    Google Scholar 

  • Wang EA, Rosen V, Alessandro JSD et al (1990) Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci 87:2220–2224

    Article  CAS  PubMed  Google Scholar 

  • Wang Y-P, Wei Z-Y, Zhong X-F et al (2015) Stable expression of basic fibroblast growth factor in chloroplasts of tobacco. Int J Mol Sci 17:19

    Article  CAS  PubMed Central  Google Scholar 

  • Yarbakht M, Jalali-Javaran M, Nikkhah M, Mohebodini M (2015) Dicistronic expression of human proinsulin-protein A fusion in tobacco chloroplast. Biotechnol Appl Biochem 62:55–63

    Article  CAS  PubMed  Google Scholar 

  • Zhang B, Shanmugaraj B, Daniell H (2017) Expression and functional evaluation of biopharmaceuticals made in plant chloroplasts. Curr Opin Chem Biol 38:17–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We are grateful to Prof. Roger N. Beachy (Danforth Center, USA) for providing the β-conglycinin regulatory sequences, Dr. Rafael A. Siqueira and Jéssica A. Ferreira for their contribution with BMP2 activity assays. This study was supported by the Empresa Brasileira de Pesquisa Agropecuária (Embrapa), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and International Association for Dental Research/Osseointegration Foundation—Implant Innovation Award.

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Authors and Affiliations

  1. Embrapa Recursos Genéticos e Biotecnologia, PqEB W5 Norte, Brasília, DF, 70770-900, Brazil

    Lídia N. Queiroz, Franciele R. Maldaner, Érica A. Mendes, Aline R. Sousa, Rebeca C. D’Allastta & Francisco J. L. Aragão

  2. Departamento de Biologia Celular, Universidade de Brasília, Campus Universitário, Brasília, DF, 70910-900, Brazil

    Lídia N. Queiroz

  3. Biological and Material Sciences, School of Dentistry, University of Michigan, 1011 North University, Ann Arbor, MI, 49109-1078, USA

    Lídia N. Queiroz, Gustavo Mendonça & Daniela B. S. Mendonça

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  1. Lídia N. Queiroz
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  2. Franciele R. Maldaner
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  3. Érica A. Mendes
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  4. Aline R. Sousa
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  8. Francisco J. L. Aragão
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Correspondence to Francisco J. L. Aragão.

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Queiroz, L.N., Maldaner, F.R., Mendes, É.A. et al. Evaluation of lettuce chloroplast and soybean cotyledon as platforms for production of functional bone morphogenetic protein 2. Transgenic Res 28, 213–224 (2019). https://doi.org/10.1007/s11248-019-00116-7

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