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Abstract:

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186 amino acids of bacterial β-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors, such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism responsible for hEGF instability in chloroplasts is regulated by light. © 2006 Elsevier B.V. All rights reserved.

Registro:

Documento: Artículo
Título:Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light
Autor:Wirth, S.; Segretin, M.E.; Mentaberry, A.; Bravo-Almonacid, F.
Filiación:Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, INGEBI-CONICET, FCEN-UBA, Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina
Palabras clave:Chloroplast transformation; Fusion protein; hEGF; Molecular farming; Transplastomic tobacco; Plants (botany); Tobacco; Chloroplast transformation; Electron transport inhibitors; Fusion protein; Human epidermal growth factor (hEGF); Molecular farming; Transplastomic tobacco; Proteins; amino acid; bacterial enzyme; beta glucuronidase; gamma urogastrone; hybrid protein; protein; urea derivative; viologen; article; chloroplast; controlled study; electron transport; genetic transcription; incubation time; light dark cycle; nonhuman; nucleotide sequence; plant; plant leaf; priority journal; protein expression; Blotting, Northern; Blotting, Southern; Blotting, Western; Chloroplasts; Epidermal Growth Factor; Gene Expression Regulation, Plant; Genetic Vectors; Glucuronidase; Humans; Light; Plants, Genetically Modified; Recombinant Fusion Proteins; Tobacco; Chlorophylls; Plants; Plastids; Proteins; Tobacco; Bacteria (microorganisms); Nicotiana obtusifolia
Año:2006
Volumen:125
Número:2
Página de inicio:159
Página de fin:172
DOI: http://dx.doi.org/10.1016/j.jbiotec.2006.02.012
Título revista:Journal of Biotechnology
Título revista abreviado:J. Biotechnol.
ISSN:01681656
CODEN:JBITD
CAS:amino acid, 65072-01-7; beta glucuronidase, 9001-45-0; gamma urogastrone, 62253-61-6; protein, 67254-75-5; Epidermal Growth Factor, 62229-50-9; Glucuronidase, EC 3.2.1.31; Recombinant Fusion Proteins
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_01681656_v125_n2_p159_Wirth

Referencias:

  • Adam, Z., Clarke, A., Cutting edge of chloroplast proteolysis (2002) Trends Plant Sci., 7, pp. 451-456
  • Balmer, Y., Koller, A., del Val, G., Manieri, W., Schürmann, P., Buchanan, B., Proteomics gives insight into the regulatory function of chloroplast thioredoxins (2003) Proc. Natl. Acad. Sci. U.S.A., 100, pp. 370-375
  • Bellucci, M., De Marchis, F., Mannucci, R., Bock, R., Arcioni, S., Cytoplasm and chloroplasts are not suitable subcellular locations for β-zein accumulation in transgenic plants (2005) J. Exp. Bot., 56, pp. 1205-1212
  • Berlanga Acosta, J., Mella Lizama, C., Some considerations on the physiological role of epidermal growth factor in relation to its pharmacological applications (1998) Biotechnol. Appl., 15, pp. 141-148
  • Birch-Machin, I., Newell, C., Hibberd, J., Gray, J., Accumulation of rotavirus VP6 protein in chloroplasts of transplastomic tobacco is limited by protein stability (2004) Plant Biotechnol. J., 2, pp. 261-270
  • Bock, R., Khan, M., Taming plastids for a green future (2004) Trends Biotechnol., 22, pp. 311-318
  • Church, G.M., Gilbert, W., Genomic sequencing (1984) Proc. Natl. Acad. Sci. U.S.A., 81, pp. 1991-1995
  • Daniell, H., Lee, S., Panchal, T., Wiebe, P., Expression of the native cholera toxin B subunit gene and assembly of functional oligomers in transgenic tobacco chloroplasts (2001) J. Mol. Biol., 311, pp. 1001-1009
  • Daniell, H., Transformation and foreign gene expression in plants by microprojectile bombardment (1997) Methods Mol. Biol., 62, pp. 463-489
  • De Cosa, B., Moar, W., Lee, S.-B., Miller, M., Daniell, H., Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals (2001) Nat. Biotechnol., 19, pp. 71-74
  • Dellaporta, S., Wood, J., Hicks, J., A plant DNA minipreparation: version II (1983) Plant Mol. Biol. Rep., 1, pp. 19-21
  • Eibl, C., Zou, Z., Beck, A., Kim, M., Mullet, J., Koop, H., In vivo analysis of plastid psbA, rbcL and rpl32 UTR elements by chloroplast transformation: tobacco plastid gene expression is controlled by modulation of transcript levels and translation efficiency (1999) Plant J., 19, pp. 333-345
  • Fernández-San Millán, A., Mingo-Castel, A., Miller, M., Daniell, H., A chloroplast transgenic approach to hyper-express and purify human serum albumin, a protein highly susceptible to proteolytic degradation (2003) Plant Biotechnol. J., 1, pp. 71-79
  • Hirose, T., Sugiura, M., Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs (2004) Plant Cell Physiol., 45, pp. 114-117
  • Kim, J., Mayfield, S., The active site of the thioredoxin-like domain of chloroplast protein disulfide isomerase, RB60, catalyzes the redox-regulated binding of chloroplast poly(A)-binding protein, RB47, to the 5′ untranslated region of psbA mRNA (2002) Plant Cell Physiol., 43, pp. 1238-1243
  • Leelavathi, S., Reddy, V., Chloroplast expression of His-tagged GUS-fusions: a general strategy to overproduce and purify foreign proteins using transplastomic plants as bioreactors (2003) Mol. Breed., 11, pp. 49-58
  • Levitan, A., Trebish, T., Kiss, V., Pereg, Y., Dangoor, I., Danon, A., Dual targeting of the protein disulfide isomerase RB60 to the chloroplast and the endoplasmic reticulum (2005) Proc. Natl. Acad. Sci. U.S.A., 102, pp. 6225-6230
  • Magee, A., Horvath, E., Kavanagh, T., Pre-screening plastid transgene expression in Escherichia coli may be unreliable as a predictor of expression levels in chloroplast-transformed plants (2004) Plant Sci., 166, pp. 1605-1611
  • Maliga, P., Plastid transformation in higher plants (2004) Annu. Rev. Plant Biol., 55, pp. 289-313
  • Palatnik, J., Carrillo, N., Valle, E., The role of photosynthetic electron transport in the oxidative degradation of chloroplastic glutamine synthetase (1999) Plant Physiol., 121, pp. 471-478
  • Schurmann, P., Jacquot, J., Plant thioredoxin systems revisited (2000) Annu. Rev. Plant Physiol. Plant Mol. Biol., 51, pp. 371-400
  • Staub, J., Garcia, B., Graves, J., Hajdukiewicz, P., Hunter, P., Nehra, N., Paradkar, V., Russell, D., High-yield production of a human therapeutic protein in tobacco chloroplasts (2000) Nat. Biotechnol., 18, pp. 333-338
  • Tanaka, R., Mizukami, M., Ishibashi, M., Tokunaga, H., Tokunaga, M., Cloning and expression of the ccdA-associated thiol-disulfide oxidoreductase (catA) gene from Brevibacillus choshinensis: stimulation of human epidermal growth factor production (2003) J. Biotechnol., 103, pp. 1-10
  • Tewari, K., Purification and properties of chloroplast DNA polymerase (1986) Methods Enzymol., 118, pp. 186-201
  • Trebitsh, T., Levitan, A., Sofer, A., Danon, A., Translation of chloroplast psbA mRNA is modulated in the light by counteracting oxidizing and reducing activities (2000) Mol. Cell. Biol., 20, pp. 1116-1123
  • Tregoning, J., Nixon, P., Kuroda, H., Svab, Z., Clare, S., Bowe, F., Fairweather, N., Maliga, P., Expression of tetanus toxin fragment C in tobacco chloroplasts (2003) Nucleic Acids Res., 31, pp. 1174-1179
  • Triboush, S., Danilenko, N., Davydenko, O., A method for isolation of chloroplast DNA and mitochondrial DNA from sunflower (1998) Plant Mol. Biol. Rep., 16, pp. 183-189
  • Tsai, B., Rodighiero, C., Lencer, W., Rapoport, T., Protein disulfide isomerase acts as a redox-dependent chaperone to unfold cholera toxin (2001) Cell, 104, pp. 937-948
  • Wirth, S., Calamante, G., Mentaberry, A., Bussmann, L., Lattanzi, M., Barañao, L., Bravo-Almonacid, F., Expression of active human epidermal growth factor (hEGF) in tobacco plants by integrative and non-integrative systems (2004) Mol. Breed., 13, pp. 23-35
  • Ye, G.-N., Hajdukiewicz, P., Broyles, D., Rodriguez, D., Xu, C., Nehra, N., Staub, J., Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco (2001) Plant J., 25, pp. 261-270
  • Zhang, Q., Liu, Y., Sodmergen, Examination of the cytoplasmic DNA in male reproductive cells to determine the potential for cytoplasmic inheritance in 295 angiosperm species (2003) Plant Cell Physiol., 44, pp. 941-951

Citas:

---------- APA ----------
Wirth, S., Segretin, M.E., 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. Journal of Biotechnology, 125(2), 159-172.
http://dx.doi.org/10.1016/j.jbiotec.2006.02.012
---------- CHICAGO ----------
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" . Journal of Biotechnology 125, no. 2 (2006) : 159-172.
http://dx.doi.org/10.1016/j.jbiotec.2006.02.012
---------- MLA ----------
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" . Journal of Biotechnology, vol. 125, no. 2, 2006, pp. 159-172.
http://dx.doi.org/10.1016/j.jbiotec.2006.02.012
---------- VANCOUVER ----------
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. J. Biotechnol. 2006;125(2):159-172.
http://dx.doi.org/10.1016/j.jbiotec.2006.02.012