Skip to main content

Advertisement

SpringerLink
Log in

Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that chloroplast expression of many proteins, such as human epidermal growth factor (hEGF), results hindered by post-transcriptional mechanisms. hEGF degradation has been related to the redox potential of the stroma and protein misfolding. To solve this problem, we proposed the redirection of hEGF into the thylakoid lumen where the environment could improve disulfide bonds formation stabilizing the functional conformation of the protein. We generated transplastomic tobacco plants targeting hEGF protein to the thylakoid lumen by adding a transit peptide (Str). Following this approach, we could detect thylakoid lumen-targeted hEGF by western blotting while stromal accumulation of hEGF remained undetectable. Southern blot analysis confirmed the integration of the transgene through homologous recombination into the plastome. Northern blot analysis showed similar levels of egf transcripts in the EGF and StrEGF lines. These results suggest that higher stability of the hEGF peptide in the thylakoid lumen is the primary cause of the increased accumulation of the recombinant protein observed in StrEGF lines. They also highlight the necessity of exploring different sub-organellar destinations to improve the accumulation levels of a specific recombinant protein in plastids.

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

Similar content being viewed by others

References

  • Abdull Razis AF, Ismail EN, Hambali Z, Abdullah MN, Ali AM, Mohd Lila MA (2008) Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity. Appl Biochem Biotechnol 144:249–261

    Article  CAS  PubMed  Google Scholar 

  • Ahmad N, Michoux F, McCarthy J, Nixon PJ (2012) Expression of the affinity tags, glutathione-S-transferase and maltose-binding protein, in tobacco chloroplasts. Planta 235:863–871

    Article  CAS  PubMed  Google Scholar 

  • Albiniak AM, Baglieri J, Robinson C (2012) Targeting of lumenal proteins across the thylakoid membrane. J Exp Bot 63:1689–1698

    Article  CAS  PubMed  Google Scholar 

  • Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H (2007) Field production and functional evaluation of chloroplast-derived interferon-alpha2b. Plant Biotechnol J 5:511–525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ayliffe MA, Scott NS, Timmis JN (1998) Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants. Mol Biol Evol 15:738–745

    Article  CAS  PubMed  Google Scholar 

  • Bally J, Nadai M, Vitel M, Rolland A, Dumain R, Dubald M (2009) Plant physiological adaptations to the massive foreign protein synthesis occurring in recombinant chloroplasts. Plant Physiol 150:1474–1481

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bock R (2014) Engineering chloroplasts for high-level foreign protein expression. Methods Mol Biol 1132:93–106

    Article  CAS  PubMed  Google Scholar 

  • Brown GL, Nanney LB, Griffen J, Cramer AB, Yancey JM, Curtsinger LJ III, Holtzin L, Schultz GS, Jurkiewicz MJ, Lynch JB (1989) Enhancement of wound healing by topical treatment with epidermal growth factor. N Engl J Med 321:76–79

    Article  CAS  PubMed  Google Scholar 

  • Burnett MJB, Burnett AC (2020) Therapeutic recombinant protein production in plants: challenges and opportunities. Plants People Planet 2:121–132

    Article  Google Scholar 

  • Corigliano MG, Albarracín RM, Vilas JM, Sánchez López EF, Bengoa Luoni SA, Deng B, Farran I, Veramendi J, Maiale SJ, Sander VA, Clemente M (2019) Heat treatment alleviates the growth and photosynthetic impairment of transplastomic plants expressing Leishmania infantum Hsp83–Toxoplasma gondii SAG1 fusion protein. Plant Sci 284:117–126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • De Cosa B, Moar W, Lee SB, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74

    Article  PubMed  PubMed Central  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  PubMed  PubMed Central  CAS  Google Scholar 

  • De Marchis F, Bellucci M, Pompa A (2016) Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation. Plant Biotechnol J 14:603–614

    Article  PubMed  CAS  Google Scholar 

  • Dellaporta SL, Chomet PS, Mottinger JP, Wood JA, Yu SM, Hicks JB (1984) Endogenous transposable elements associated with virus infection in maize. Cold Spring Harb Symp Quant Biol 49:321–328

    Article  CAS  PubMed  Google Scholar 

  • Du J-J, Zhan C-Y, Lu Y, Cui H-R, Wang X-Y (2015) The conservative cysteines in transmembrane domain of AtVKOR/LTO1 are critical for photosynthetic growth and photosystem II activity in Arabidopsis. Front Plant Sci 6:238

    PubMed  PubMed Central  Google Scholar 

  • Fernandez-San Millan A, Mingo-Castel A, Miller M, Daniell H (2003) A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation. Plant Biotechnol J 1:71–79

    Article  CAS  PubMed  Google Scholar 

  • Giddings G, Allison G, Brooks D, Carter A (2000) Transgenic plants as factories for biopharmaceuticals. Nat Biotechnol 18:1151–1155

    Article  CAS  PubMed  Google Scholar 

  • Giorgi C, Franconi R, Rybicki EP (2010) Human papillomavirus vaccines in plants. Expert Rev Vaccines 9:913–924

    Article  CAS  PubMed  Google Scholar 

  • Gundinger T, Spadiut O (2017) A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli. J Biotechnol 248:15–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Habibi P, Daniell H, Soccol CR, Grossi-de-Sa MF (2019) The potential of plant systems to break the HIV-TB link. Plant Biotechnol J 17:1868–1891

    Article  PubMed  PubMed Central  Google Scholar 

  • Kondo A, Kohda J, Endo Y, Shiromizu T, Kurokawa Y, Nishihara K, Yanagi H, Yura T, Fukuda H (2000) Improvement of productivity of active horseradish peroxidase in Escherichia coli by coexpression of Dsb proteins. J Biosci Bioeng 90:600–606

    Article  CAS  PubMed  Google Scholar 

  • Kwon KC, Nityanandam R, New JS, Daniell H (2013) Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells. Plant Biotechnol J 11:77–86

    Article  CAS  PubMed  Google Scholar 

  • Lentz EM, Segretin ME, Morgenfeld MM, Wirth SA, Dus Santos MJ, Mozgovoj MV, Wigdorovitz A, Bravo-Almonacid FF (2010) High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants. Planta 231:387–395

    Article  CAS  PubMed  Google Scholar 

  • Lentz EM, Garaicoechea L, Alfano EF, Parreno V, Wigdorovitz A, Bravo-Almonacid FF (2012) Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco. Planta 236:703–714

    Article  CAS  PubMed  Google Scholar 

  • Lossl A, Eibl C, Harloff HJ, Jung C, Koop HU (2003) Polyester synthesis in transplastomic tobacco (Nicotiana tabacum L.): significant contents of polyhydroxybutyrate are associated with growth reduction. Plant Cell Rep 21:891–899

    Article  CAS  PubMed  Google Scholar 

  • Maliga P, Bock R (2011) Plastid biotechnology: food, fuel, and medicine for the 21st century. Plant Physiol 155:1501–1510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marques JP, Schattat MH, Hause G, Dudeck I, Klosgen RB (2004) In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana. J Exp Bot 55:1697–1706

    Article  CAS  PubMed  Google Scholar 

  • Maskos K, Huber-Wunderlich M, Glockshuber R (2003) DsbA and DsbC-catalyzed oxidative folding of proteins with complex disulfide bridge patterns in vitro and in vivo. J Mol Biol 325:495–513

    Article  CAS  PubMed  Google Scholar 

  • Morgenfeld M, Lentz E, Segretin ME, Alfano EF, Bravo-Almonacid F (2014) Translational fusion and redirection to thylakoid lumen as strategies to enhance accumulation of human papillomavirus E7 antigen in tobacco chloroplasts. Mol Biotechnol 56:1021–1031

    Article  CAS  PubMed  Google Scholar 

  • Nakazono M, Hirai A (1993) Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice. Mol Gen Genet 236:341–346

    Article  CAS  PubMed  Google Scholar 

  • Obembe OO, Popoola JO, Leelavathi S, Reddy SV (2011) Advances in plant molecular farming. Biotechnol Adv 29:210–222

    Article  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 

  • Rigano MM, Manna C, Giulini A, Pedrazzini E, Capobianchi M, Castilletti C, Di Caro A, Ippolito G, Beggio P, De Giuli MC, Monti L, Vitale A, Cardi T (2009) Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cellsdagger. Plant Biotechnol J 7:577–591

    Article  CAS  PubMed  Google Scholar 

  • Ruf S, Biehler K, Bock R (2000) A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna. J Cell Biol 149:369–378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ruhlman T, Ahangari R, Devine A, Samsam M, Daniell H (2007) Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts–oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J 5:495–510

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schlapschy M, Skerra A (2011) Periplasmic chaperones used to enhance functional secretion of proteins in E. coli. Methods Mol Biol 705:211–224

    Article  CAS  PubMed  Google Scholar 

  • Scotti N, Cardi T (2014) Transgene-induced pleiotropic effects in transplastomic plants. Biotechnol Lett 36:229–239

    Article  CAS  PubMed  Google Scholar 

  • Scotti N, Alagna F, Ferraiolo E, Formisano G, Sannino L, Buonaguro L, De Stradis A, Vitale A, Monti L, Grillo S, Buonaguro FM, Cardi T (2009) High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts. Planta 229:1109–1122

    Article  CAS  PubMed  Google Scholar 

  • Scotti N, Rigano MM, Cardi T (2012) Production of foreign proteins using plastid transformation. Biotechnol Adv 30:387–397

    Article  CAS  PubMed  Google Scholar 

  • Sparrow PA, Twyman RM (2009) Biosafety, risk assessment and regulation of plant-made pharmaceuticals. Methods Mol Biol 483:341–353

    Article  PubMed  Google Scholar 

  • Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V, Schlittler M, Carroll JA, Spatola L, Ward D, Ye G, Russell DA (2000) High-yield production of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol 18:333–338

    Article  CAS  PubMed  Google Scholar 

  • Stern DB, Lonsdale DM (1982) Mitochondrial and chloroplast genomes of maize have a 12-kilobase DNA sequence in common. Nature 299:698–702

    Article  CAS  PubMed  Google Scholar 

  • Stern DB, Goldschmidt-Clermont M, Hanson MR (2010) Chloroplast RNA metabolism. Annu Rev Plant Biol 61:125–155

    Article  CAS  PubMed  Google Scholar 

  • Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87:8526–8530

    Article  CAS  PubMed  Google Scholar 

  • Tanaka R, Hashimoto M, Ishibashi M, Tokunaga H, Taura S, Mizukami M, Miyauchi A, Tokunaga M (2001) Secretion of Escherichia coli DsbA and DsbC proteins from Brevibacillus choshinensis: stimulation of human epidermal growth factor production. Biotechnol Lett 23:1853–1857

    Article  CAS  Google Scholar 

  • Tong WY, Yao SJ, Zhu ZQ, Yu J (2001) An improved procedure for production of human epidermal growth factor from recombinant E. coli. Appl Microbiol Biotechnol 57:674–679

    Article  CAS  PubMed  Google Scholar 

  • van Eerde A, Gottschamel J, Bock R, Hansen KEA, Munang’andu HM, Daniell H, Liu Clarke J (2019) Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development. Plant Biotechnol J 17:1408–1417

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wang X, Brandsma M, Tremblay R, Maxwell D, Jevnikar AM, Huner N, Ma S (2008) A novel expression platform for the production of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65). BMC Biotechnol 8:87

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wirth S, Segretin ME, Mentaberry A, Bravo-Almonacid F (2006) Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light. J Biotechnol 125:159–172

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Dr. María Eugenia Segretin and Lic. Federico Gabriel Mirkin for critical reading of this manuscript. This work has been supported by Grant PICT 2014-2766 from Agencia Nacional de Promoción Científica y Tecnológica. EFA and NB are fellows of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina). MM and FBA are research scientists of CONICET. This work has also been supported by Fondo para la Investigación Científica y Tecnológica (Grant No. 2013-1355).

Author information

Authors and Affiliations

  1. Instituto de Ingeniería Genética y Biología Molecular “Dr, Héctor Torres” (INGEBI-CONICET), Vuelta de Obligado 2490, Ciudad Autónoma de Buenos Aires, Argentina

    Mauro M. Morgenfeld, Catalina F. Vater, E. Federico Alfano, Noelia A. Boccardo & Fernando F. Bravo-Almonacid

  2. Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina

    Fernando F. Bravo-Almonacid

  3. Departamento de Fisiología, Biología Molecular y Celular (FCEN-UBA), Buenos Aires, Argentina

    Mauro M. Morgenfeld

Authors
  1. Mauro M. Morgenfeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Catalina F. Vater
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E. Federico Alfano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Noelia A. Boccardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fernando F. Bravo-Almonacid
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

FBA and MM conceived and designed research. FBA directed the project. MM, NB, CV, and EFA conducted experiments. FBA and MM analyzed data. FBA and MM wrote the manuscript. All authors read and approved the manuscript.

Corresponding author

Correspondence to Fernando F. Bravo-Almonacid.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Morgenfeld, M.M., Vater, C.F., Alfano, E.F. et al. Translocation from the chloroplast stroma into the thylakoid lumen allows expression of recombinant epidermal growth factor in transplastomic tobacco plants. Transgenic Res 29, 295–305 (2020). https://doi.org/10.1007/s11248-020-00199-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-020-00199-7

Keywords

Search

Navigation