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Human papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death in women worldwide, and its E7 antigen is the major candidate for a therapeutic vaccine. The large scale production of E7 by molecular farming that would lead to the development of a safe and inexpensive vaccine is impaired by its low accumulation level in the plant cell. To enhance antigen production in the plastids, two alternative strategies were carried out: the expression of E7 as a translational fusion to β-glucuronidase enzyme and redirection of E7 into the thylakoid lumen. The use of the β-glucuronidase as a partner protein turned out to be a successful strategy, antigen expression levels were enhanced between 30 and 40 times relative to unfused E7. Moreover, best accumulation, albeit at a high metabolic cost that compromised biomass production, was obtained redirecting E7 into the thylakoid lumen by the incorporation of the N-terminal transit peptide, Str. Following this approach lumenal E7 production exceeded the stromal by two orders of magnitude. Our results highlight the relevance of exploring different strategies to improve recombinant protein stability for certain transgenes in order to exploit potential advantages of recombinant protein accumulation in chloroplasts. © 2014, Springer Science+Business Media New York.


Documento: Artículo
Título:Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts
Autor:Morgenfeld, M.; Lentz, E.; Segretin, M.E.; Alfano, E.F.; Bravo-Almonacid, F.
Filiación:Instituto de Ingeniería Genética y Biología Molecular “Dr, Hector Torres” (INGEBI-CONICET), Vuelta de Obligado 2490, C.C 1428ADN, Ciudad Autónoma de Buenos Aires, Argentina
Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Bernal, Buenos Aires, Argentina
Departamento de Fisiología, Biología Molecular y Celular (FCEN-UBA), Intendente Güiraldes 2620 Pab. II, C.I. 428EHA, Ciudad Universitaria, Buenos Aires, Argentina
Palabras clave:E7 antigen; Fusion protein; Human papillomavirus; Molecular farming; Thylakoid translocation; Transplastomic tobacco; Antigens; Oncogenic viruses; Plants (botany); Tobacco; Vaccines; Fusion proteins; Human papillomavirus; Large scale productions; Molecular farming; Protein accumulation; Therapeutic vaccines; Thylakoids; Translational fusion; Recombinant proteins; beta glucuronidase; hybrid protein; oncogene protein E7, Human papillomavirus type 16; protein E7; chloroplast; genetics; growth, development and aging; metabolism; molecular cloning; phenotype; thylakoid; tobacco; transgenic plant; Chloroplasts; Cloning, Molecular; Glucuronidase; Papillomavirus E7 Proteins; Phenotype; Plants, Genetically Modified; Recombinant Fusion Proteins; Thylakoids; Tobacco
Página de inicio:1021
Página de fin:1031
Título revista:Molecular Biotechnology
Título revista abreviado:Mol. Biotechnol.
CAS:beta glucuronidase, 9001-45-0; Glucuronidase; oncogene protein E7, Human papillomavirus type 16; Papillomavirus E7 Proteins; Recombinant Fusion Proteins


  • Sharma, A.K., Sharma, M.K., Plants as bioreactors: Recent developments and emerging opportunities (2009) Biotechnology Advances, 27, pp. 811-832. , COI: 1:CAS:528:DC%2BD1MXht1CqtbnJ
  • Obembe, O.O., Popoola, J.O., Leelavathi, S., Reddy, S.V., Advances in plant molecular farming (2011) Biotechnology Advances, 29, pp. 210-222
  • Maliga, P., Bock, R., Plastid biotechnology: Food, fuel, and medicine for the 21st century (2011) Plant Physiology, 155, pp. 1501-1510. , COI: 1:CAS:528:DC%2BC3MXkvVOrsL0%3D
  • Scotti, N., Rigano, M.M., Cardi, T., Production of foreign proteins using plastid transformation (2012) Biotechnology Advances, 30, pp. 387-397. , COI: 1:CAS:528:DC%2BC38Xit1aqtbg%3D
  • Daniell, H., Production of biopharmaceuticals and vaccines in plants via the chloroplast genome (2006) Biotechnology Journal, 1, pp. 1071-1079. , COI: 1:CAS:528:DC%2BD2sXisVyksr4%3D
  • Bock, R., Khan, M.S., Taming plastids for a green future (2004) Trends in Biotechnology, 22, pp. 311-318. , COI: 1:CAS:528:DC%2BD2cXkt1agsbk%3D
  • Maliga, P., Plastid transformation in higher plants (2004) Annual Review of Plant Biology, 55, pp. 289-313. , COI: 1:CAS:528:DC%2BD2cXlvFeisL4%3D
  • Sakamoto, W., Protein degradation machineries in plastids (2006) Annual Review of Plant Biology, 57, pp. 599-621. , COI: 1:CAS:528:DC%2BD28XosVKht7o%3D
  • Castellsague, X., Natural history and epidemiology of HPV infection and cervical cancer (2008) Gynecologic Oncology, 110, pp. S4-S7
  • Van Kriekinge, G., Castellsague, X., Cibula, D., Demarteau, N., Estimation of the potential overall impact of human papillomavirus vaccination on cervical cancer cases and deaths (2014) Vaccine, 32, pp. 733-739
  • Koutsky, L.A., Ault, K.A., Wheeler, C.M., Brown, D.R., Barr, E., Alvarez, F.B., Chiacchierini, L.M., for the Proof of Principle Study Investigators, A controlled trial of a human papillomavirus type 16 vaccine (2002) New England Journal of Medicine, 347, pp. 1645-1651. , COI: 1:CAS:528:DC%2BD38XovFSjurs%3D
  • Paolini, F., Massa, S., Manni, I., Franconi, R., Venuti, A., Immunotherapy in new pre-clinical models of HPV-associated oral cancers (2013) Human Vaccines & Immunotherapeutics, 9, pp. 534-543. , COI: 1:CAS:528:DC%2BC3sXhvVOgu7fF
  • Hallez, S., Simon, P., Maudoux, F., Doyen, J., Noel, J.C., Beliard, A., Capelle, X., Jacobs, N., Phase I/II trial of immunogenicity of a human papillomavirus (HPV) type 16 E7 protein-based vaccine in women with oncogenic HPV-positive cervical intraepithelial neoplasia (2004) Cancer Immunology, Immunotherapy, 53, pp. 642-650. , COI: 1:CAS:528:DC%2BD2cXksFSit7Y%3D
  • Trimble, C.L., Peng, S., Kos, F., Gravitt, P., Viscidi, R., Sugar, E., Pardoll, D., Wu, T.C., A phase I trial of a human papillomavirus DNA vaccine for HPV16+ cervical intraepithelial neoplasia 2/3 (2009) Clinical Cancer Research, 15, pp. 361-367. , COI: 1:CAS:528:DC%2BD1MXhs1eqtA%3D%3D
  • Smotkin, D., Wettstein, F.O., The major human papillomavirus protein in cervical cancers is a cytoplasmic phosphoprotein (1987) Journal of Virology, 61, pp. 1686-1689. , COI: 1:CAS:528:DyaL2sXkt1SnsLo%3D
  • Alonso, L.G., Garcia-Alai, M.M., Nadra, A.D., Lapena, A.N., Almeida, F.L., Gualfetti, P., Prat-Gay, G.D., High-risk (HPV16) human papillomavirus E7 oncoprotein is highly stable and extended, with conformational transitions that could explain its multiple cellular binding partners (2002) Biochemistry, 41, pp. 10510-10518. , COI: 1:CAS:528:DC%2BD38XlsFamtLc%3D
  • Morgenfeld, M., Segretin, M.E., Wirth, S., Lentz, E., Zelada, A., Mentaberry, A., Gissmann, L., Bravo-Almonacid, F., Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast (2009) Molecular Biotechnology, 43, pp. 243-249. , COI: 1:CAS:528:DC%2BD1MXht1Cmtb3M
  • Demurtas, O.C., Massa, S., Ferrante, P., Venuti, A., Franconi, R., Giuliano, G., A Chlamydomonas-derived Human Papillomavirus 16 E7 vaccine induces specific tumor protection (2013) PLoS ONE, 8, p. e61473. , COI: 1:CAS:528:DC%2BC3sXntVKrsbs%3D
  • Uversky, V.N., Roman, A., Oldfield, C.J., Dunker, A.K., Protein intrinsic disorder and human papillomaviruses: Increased amount of disorder in E6 and E7 oncoproteins from high risk HPVs (2006) Journal of Proteome Research, 5, pp. 1829-1842. , COI: 1:CAS:528:DC%2BD28Xms1eqtbY%3D
  • Staub, J.M., Garcia, B., Graves, J., Hajdukiewicz, P.T., Hunter, P., Nehra, N., Paradkar, V., Russell, D.A., High-yield production of a human therapeutic protein in tobacco chloroplasts (2000) Nature Biotechnology, 18, pp. 333-338. , COI: 1:CAS:528:DC%2BD3cXhvVyqsr0%3D
  • Ye, G.N., Hajdukiewicz, P.T., Broyles, D., Rodriguez, D., Xu, C.W., Nehra, N., Staub, J.M., Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco (2001) The Plant Journal, 25, pp. 261-270. , COI: 1:CAS:528:DC%2BD3MXitlKnu7w%3D
  • Mishra, S., Yadav, D.K., Tuli, R., Ubiquitin fusion enhances cholera toxin B subunit expression in transgenic plants and the plant-expressed protein binds GM1 receptors more efficiently (2006) Journal of Biotechnology, 127, pp. 95-108. , COI: 1:CAS:528:DC%2BD28Xht1ShtLzJ
  • Elghabi, Z., Karcher, D., Zhou, F., Ruf, S., Bock, R., Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome (2011) Plant Biotechnology Journal, 9, pp. 599-608. , COI: 1:CAS:528:DC%2BC3MXotFyksrY%3D
  • Wirth, S., Segretin, M.E., Mentaberry, A., Bravo-Almonacid, F., Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light (2006) Journal of Biotechnology, 125, pp. 159-172. , COI: 1:CAS:528:DC%2BD28Xot1Wkt7o%3D
  • Schurmann, P., Jacquot, J.P., Plant thioredoxin systems revisited (2000) Annual Review of Plant Physiology and Plant Molecular Biology, 51, pp. 371-400. , COI: 1:CAS:528:DC%2BD3cXlsVymt7w%3D
  • Reddie, K.G., Carroll, K.S., Expanding the functional diversity of proteins through cysteine oxidation (2008) Current Opinion in Chemical Biology, 12, pp. 746-754. , COI: 1:CAS:528:DC%2BD1cXhsFajs7zP
  • Conway, M.J., Cruz, L., Alam, S., Christensen, N.D., Meyers, C., Differentiation-dependent interpentameric disulfide bond stabilizes native human papillomavirus type 16 (2011) PLoS ONE, 6, p. e22427. , COI: 1:CAS:528:DC%2BC3MXhtVehtLjI
  • Marques, J.P., Dudeck, I., Klosgen, R.B., Targeting of EGFP chimeras within chloroplasts (2003) Molecular Genetics and Genomics, 269, pp. 381-387. , COI: 1:CAS:528:DC%2BD3sXkvF2gtbg%3D
  • Albiniak, A.M., Baglieri, J., Robinson, C., Targeting of lumenal proteins across the thylakoid membrane (2012) Journal of Experimental Botany, 63, pp. 1689-1698. , COI: 1:CAS:528:DC%2BC38XjtlKhsLo%3D
  • Jefferson, R.A., Kavanagh, T.A., Bevan, M.W., GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants (1987) EMBO Journal, 6, pp. 3901-3907. , COI: 1:CAS:528:DyaL1cXovV2itQ%3D%3D
  • Svab, Z., Hajdukiewicz, P., Maliga, P., Stable transformation of plastids in higher plants (1990) Proceedings of the National Academy of Sciences of the United States of America, 87, pp. 8526-8530. , COI: 1:CAS:528:DyaK3MXhslWn
  • Dellaporta, S.L., Chomet, P.S., Mottinger, J.P., Wood, J.A., Yu, S.M., Hicks, J.B., Endogenous transposable elements associated with virus infection in maize (1984) Cold Spring Harbor Symposia on Quantitative Biology, 49, pp. 321-328. , COI: 1:CAS:528:DyaL28XmtlWrsA%3D%3D
  • Church, G.M., Gilbert, W., Genomic sequencing (1984) Proceedings of the National Academy of Sciences of the United States of America, 81, pp. 1991-1995. , COI: 1:CAS:528:DyaL2cXkt1Sit7k%3D
  • Ruf, S., Biehler, K., Bock, R., A small chloroplast-encoded protein as a novel architectural component of the light-harvesting antenna (2000) Journal of Cell Biology, 149, pp. 369-378. , COI: 1:CAS:528:DC%2BD3cXislKqsLY%3D
  • Ayliffe, M.A., Scott, N.S., Timmis, J.N., Analysis of plastid DNA-like sequences within the nuclear genomes of higher plants (1998) Molecular Biology and Evolution, 15, pp. 738-745. , COI: 1:CAS:528:DyaK1cXjsFShtrY%3D
  • Nakazono, M., Hirai, A., Identification of the entire set of transferred chloroplast DNA sequences in the mitochondrial genome of rice (1993) Molecular and General Genetics, 236, pp. 341-346. , COI: 1:CAS:528:DyaK3sXks1Cntbw%3D
  • Stern, D.B., Lonsdale, D.M., Mitochondrial and chloroplast genomes of maize have a 12-kilobase DNA sequence in common (1982) Nature, 299, pp. 698-702. , COI: 1:CAS:528:DyaL3sXht1Sntbo%3D
  • Rogalski, M., Ruf, S., Bock, R., Tobacco plastid ribosomal protein S18 is essential for cell survival (2006) Nucleic Acids Research, 34, pp. 4537-4545. , COI: 1:CAS:528:DC%2BD28XhtVygu7bL
  • Giorgi, C., Franconi, R., Rybicki, E.P., Human papillomavirus vaccines in plants (2010) Expert Review of Vaccines, 9, pp. 913-924. , COI: 1:CAS:528:DC%2BC3cXpsF2lu70%3D
  • De Marchis, F., Pompa, A., Bellucci, M., Plastid proteostasis and heterologous protein accumulation in transplastomic plants (2012) Plant Physiology, 160, pp. 571-581
  • Chemes, L.B., Glavina, J., Alonso, L.G., Marino-Buslje, C., de Prat-Gay, G., Sanchez, I.E., Sequence evolution of the intrinsically disordered and globular domains of a model viral oncoprotein (2012) PLoS ONE, 7, p. e47661. , COI: 1:CAS:528:DC%2BC38XhslWjs7vM
  • van Rooijen, G.J., Moloney, M.M., Plant seed oil-bodies as carriers for foreign proteins (1995) Biotechnology (New York), 13, pp. 72-77
  • Hondred, D., Walker, J.M., Mathews, D.E., Vierstra, R.D., Use of ubiquitin fusions to augment protein expression in transgenic plants (1999) Plant Physiology, 119, pp. 713-724. , COI: 1:CAS:528:DyaK1MXhsFSksL4%3D
  • Patel, J., Zhu, H., Menassa, R., Gyenis, L., Richman, A., Brandle, J., Elastin-like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco leaves (2007) Transgenic Research, 16, pp. 239-249. , COI: 1:CAS:528:DC%2BD2sXjt1Sqtrc%3D
  • Herz, S., Fussl, M., Steiger, S., Koop, H.U., Development of novel types of plastid transformation vectors and evaluation of factors controlling expression (2005) Transgenic Research, 14, pp. 969-982. , COI: 1:CAS:528:DC%2BD2MXht1Glt7rN
  • Gray, B.N., Ahner, B.A., Hanson, M.R., High-level bacterial cellulase accumulation in chloroplast-transformed tobacco mediated by downstream box fusions (2009) Biotechnology and Bioengineering, 102, pp. 1045-1054. , COI: 1:CAS:528:DC%2BD1MXhvFGhsr8%3D
  • Lentz, E.M., Garaicoechea, L., Alfano, E.F., Parreno, V., Wigdorovitz, A., Bravo-Almonacid, F.F., Translational fusion and redirection to thylakoid lumen as strategies to improve the accumulation of a camelid antibody fragment in transplastomic tobacco (2012) Planta, 236, pp. 703-714. , COI: 1:CAS:528:DC%2BC38XhtVyks7jP
  • Segretin, M.E., Lentz, E.M., Wirth, S.A., Morgenfeld, M.M., Bravo-Almonacid, F.F., Transformation of Solanum tuberosum plastids allows high expression levels of beta-glucuronidase both in leaves and microtubers developed in vitro (2012) Planta, 235, pp. 807-818. , COI: 1:CAS:528:DC%2BC38XksFWnurg%3D
  • Lentz, E.M., Segretin, M.E., Morgenfeld, M.M., Wirth, S.A., Dus Santos, M.J., Mozgovoj, M.V., Wigdorovitz, A., Bravo-Almonacid, F.F., High expression level of a foot and mouth disease virus epitope in tobacco transplastomic plants (2010) Planta, 231, pp. 387-395. , COI: 1:CAS:528:DC%2BC3cXisVSnsQ%3D%3D
  • Marques, J.P., Schattat, M.H., Hause, G., Dudeck, I., Klosgen, R.B., In vivo transport of folded EGFP by the DeltapH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana (2004) Journal of Experimental Botany, 55, pp. 1697-1706. , COI: 1:CAS:528:DC%2BD2cXntValt74%3D
  • Scotti, N., Cardi, T., Transgene-induced pleiotropic effects in transplastomic plants (2014) Biotechnology Letters, 36, pp. 229-239. , COI: 1:CAS:528:DC%2BC3sXhsFyntbfN
  • Hennig, A., Bonfig, K., Roitsch, T., Warzecha, H., Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis (2007) FEBS Journal, 274, pp. 5749-5758. , COI: 1:CAS:528:DC%2BD2sXhtlSksrbP
  • Oey, M., Lohse, M., Kreikemeyer, B., Bock, R., Exhaustion of the chloroplast protein synthesis capacity by massive expression of a highly stable protein antibiotic (2009) The Plant Journal, 57, pp. 436-445. , COI: 1:CAS:528:DC%2BD1MXit1Kitb8%3D
  • Bally, J., Nadai, M., Vitel, M., Rolland, A., Dumain, R., Dubald, M., Plant physiological adaptations to the massive foreign protein synthesis occurring in recombinant chloroplasts (2009) Plant Physiology, 150, pp. 1474-1481. , COI: 1:CAS:528:DC%2BD1MXovFerurg%3D


---------- APA ----------
Morgenfeld, M., Lentz, E., Segretin, M.E., Alfano, E.F. & Bravo-Almonacid, F. (2014) . Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts. Molecular Biotechnology, 56(11), 1021-1031.
---------- CHICAGO ----------
Morgenfeld, M., Lentz, E., Segretin, M.E., Alfano, E.F., Bravo-Almonacid, F. "Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts" . Molecular Biotechnology 56, no. 11 (2014) : 1021-1031.
---------- MLA ----------
Morgenfeld, M., Lentz, E., Segretin, M.E., Alfano, E.F., Bravo-Almonacid, F. "Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts" . Molecular Biotechnology, vol. 56, no. 11, 2014, pp. 1021-1031.
---------- VANCOUVER ----------
Morgenfeld, M., Lentz, E., Segretin, M.E., Alfano, E.F., Bravo-Almonacid, F. Translational Fusion and Redirection to Thylakoid Lumen as Strategies to Enhance Accumulation of Human Papillomavirus E7 Antigen in Tobacco Chloroplasts. Mol. Biotechnol. 2014;56(11):1021-1031.