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:: Volume 9, Issue 2 (2023) ::
pgr 2023, 9(2): 1-14 Back to browse issues page
Homoplasmic Stability and Cytoplasmic Inheritence of DARPin G3 Scaffold Protein in Generative and Vegetative Propagation of Transplastoic Tobacco Plants
Maryam Ehsasatvatan , Bahram Baghban Kohnehrouz *
Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran , bahrambaghban.kr@tabrizu.ac.ir
Abstract:   (3832 Views)
Plastid engineering gives numerous benefits for the next generation of transgenic technology, consisting of the convenient use of transgene stacking and the production of high expression levels of recombinant proteins. Designed ankyrin repeat proteins (DARPin) are relatively small non-immunoglobulin scaffold proteins that bind to their specific target with high affinity. The G3 is a type of DARPin designed to bind to the HER2 tyrosine kinase receptor (human epidermal growth factor receptor 2). We previously developed a bioprocess for the production of DARPin G3 in tobacco chloroplasts as an imaging agent in HER2 over-expressed cancers. In this study, we analyzed the expression and homoplasmic stability of DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants. The presence of DARPin G3 gene in the next generation of transplastomic plants was confirmed with specific primers by PCR analysis. Southern blot analysis confirmed the homoplasmic status of transplastomic plants. The western blot analysis confirmed the accumulation of the DARPin G3 in the chloroplasts of next generation of transplastomic plants. The DARPin G3 protein content was estimated around 33% by ELISA in chloroplast total soluble protein (TSP) of the transplastomic plants. Results confirmed that the DARPin G3 gene in vegetative and generative T1 generation of transplastomic tobacco plants was stably and highly expressed.
Keywords: Chloroplast transformation, DARPin G3, Homoplasmy, Transplastomic, Tobacco
Full-Text [PDF 692 kb]   (1275 Downloads)    
Type of Study: Research | Subject: Genetic engineering
References
1. Adem, M., Beyene, D. and Feyissa, T. (2017). Recent achievements obtained by chloroplast transformation. Plant Methods, 13: 30. [DOI:10.1186/s13007-017-0179-1] [PMID] [PMCID]
2. Boersma, Y.L. and Plückthun, A. (2011). DARPins and other repeat protein scaffolds: advances in engineering and applications. Current Opinion in Biotechnology, 22: 849-857. [DOI:10.1016/j.copbio.2011.06.004] [PMID]
3. Bork, P. (1993). Hundreds of ankyrin‐like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins: Structure, Function, and Bioinformatics, 17: 363-374. [DOI:10.1002/prot.340170405] [PMID]
4. Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72: 248-254. [DOI:10.1016/0003-2697(76)90527-3] [PMID]
5. Doyle, J.J. and Doyle, J.L. (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 19: 11-15.
6. Ehsasatvatan, M., Kohnehrouz, B.B., Gholizadeh, A., Ofoghi, H. and Shanehbandi, D. (2022a). The production of the first Functional antibody mimetic in higher plants: the chloroplast makes the DARPin G3 for HER2 imaging in oncology. Biological Research, 55: 31. [DOI:10.1186/s40659-022-00400-7] [PMID] [PMCID]
7. Ehsasatvatan, M., Kohnehrouz, B.B., Gholizadeh, A., Ofoghi, H. and Shanehbandi, D. (2022b). Physical and biologically effective parameters in developing transplastomic tobacco plants by particle bombardment method using PDS-1000/He. Genetic Engineering and Biosafety Journal, 10: 237-252 (In Persian).
8. Epa, V.C., Dolezal, O., Doughty, L., Xiao, X., Jost, C., Plückthun, A. and Adams, T.E. (2013). Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2. PLoS One, 8: 59163. [DOI:10.1371/journal.pone.0059163] [PMID] [PMCID]
9. Farran, I., McCarthy-Suárez, I., Río-Manterola, F., Mansilla, C., Lasarte, J.J. and Mingo-Castel, Á.M. (2010). The vaccine adjuvant extra domain A from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts. Planta, 231: 977-990. [DOI:10.1007/s00425-010-1102-4] [PMID]
10. Felberbaum, R.S. (2015). The baculovirus expression vector system: A commercial manufacturing platform for viral vaccines and gene therapy vectors. Biotechnology Journal, 10: 702-714. [DOI:10.1002/biot.201400438] [PMID] [PMCID]
11. Gharelo, R.S., Oliaei, E.D., Bandehagh, A., Khodadadi, E. and Noparvar, P.M. (2016). Production of therapeutic proteins through plant tissue and cell culture. Journal of BioScience & Biotechnology, 5: 93-104.
12. Goldstein, R., Sosabowski, J., Livanos, M., Leyton, J., Vigor, K., Bhavsar, G., Nagy-Davidescu, G., Rashid, M., Miranda, E. and Yeung, J. (2015). Development of the designed ankyrin repeat protein (DARPin) G3 for HER2 molecular imaging. European Journal of Nuclear Medicine and Molecular Imaging, 42: 288-301. [DOI:10.1007/s00259-014-2940-2] [PMID] [PMCID]
13. Holtz, B.R., Berquist, B.R., Bennett, L.D., Kommineni, V.J., Munigunti, R.K., White, E.L., Wilkerson, D.C., Wong, K.Y.I., Ly, L.H. and Marcel, S. (2015). Commercial‐scale biotherapeutics manufacturing facility for plant‐made pharmaceuticals. Plant Biotechnology Journal, 13: 1180-1190. [DOI:10.1111/pbi.12469] [PMID]
14. Kummer, L., Parizek, P., Rube, P., Millgramm, B., Prinz, A., Mittl, P.R., Kaufholz, M., Zimmermann, B., Herberg, F.W. and Plückthun, A. (2012). Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries. Proceedings of the National Academy of Sciences, 109: 2248-2257. [DOI:10.1073/pnas.1205399109] [PMID] [PMCID]
15. Laemmli, U. (1979). Slab gel electrophoresis: SDS-PAGE with discontinuous buffers. Nature, 227: 680-685. [DOI:10.1038/227680a0] [PMID]
16. Li, J., Mahajan, A. and Tsai, M.D. (2006). Ankyrin repeat: a unique motif mediating protein− protein interactions. Biochemistry, 45: 15168-15178. [DOI:10.1021/bi062188q] [PMID]
17. Livanos, M. (2019). Production of DARPins in the Yeast Pichia pastoris. Ph.D. Thesis, University College London, London, England.
18. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15: 473-497. [DOI:10.1111/j.1399-3054.1962.tb08052.x]
19. Oey, M., Lohse, M., Scharff, L.B., Kreikemeyer, B. and Bock, R. (2009). Plastid production of protein antibiotics against pneumonia via a new strategy for high-level expression of antimicrobial proteins. Proceedings of the National Academy of Sciences, 106: 6579-6584. [DOI:10.1073/pnas.0813146106] [PMID] [PMCID]
20. Parizek, P., Kummer, L., Rube, P., Prinz, A., Herberg, F.W. and Plückthun, A. (2012). Designed ankyrin repeat proteins (DARPins) as novel isoform-specific intracellular inhibitors of c-Jun N-terminal kinases. ACS Chemical Biology, 7: 1356-1366. [DOI:10.1021/cb3001167] [PMID]
21. Reddy, V.S., Leelavathi, S., Selvapandiyan, A., Raman, R., Giovanni, F., Shukla, V. and Bhatnagar, R.K. (2002). Analysis of chloroplast transformed tobacco plants with cry1Ia5 under rice psbA transcriptional elements reveal high level expression of Bt toxin without imposing yield penalty and stable inheritance of transplastome. Molecular Breeding, 9: 259-269. [DOI:10.1023/A:1020357729437]
22. Rezaei, A., Jalali-Javaran, M. and AbdoliNasab, M. (2014). Homoplasmy stability of transplastomic tobacco plants (Nicotiana tabacum CV. Xhanti) containing human tissue-type plasminogen activator (K2S form) gene in T1 generation. International Journal of Biosciences (IJB), 4: 116-125. [DOI:10.12692/ijb/4.9.116-125]
23. Rosales-Mendoza, S., Soria-Guerra, R.E., Moreno-Fierros, L., Alpuche-Solís, Á.G., Martínez-González, L. and Korban, S.S. (2010). Expression of an immunogenic F1-V fusion protein in lettuce as a plant-based vaccine against plague. Planta, 232: 409-416. [DOI:10.1007/s00425-010-1176-z] [PMID]
24. Sahshorpour, M., Amani, J., Jafari, M. and Salmanian, A.H. (2014). Expression of recombinant chimeric EspA-intimin protein in Nicotiana tobaccum for oral vaccine development. Plant Genetic Researches, 1(1): 77-94 (In Persian). [DOI:10.29252/pgr.1.1.77]
25. Scotti, N., Bellucci, M. and Cardi, T. (2013). The Chloroplasts as Platform for Recombinant Proteins Production. In: Duchene, A.M., Ed., Translation in mitochondria and other Organelles, pp. 225-262. Springer Berlin, Heidelberg, DE. [DOI:10.1007/978-3-642-39426-3_10]
26. Scotti, N., Rigano, M.M. and Cardi, T. (2012). Production of foreign proteins using plastid transformation. Biotechnology Advances, 30: 387-397. [DOI:10.1016/j.biotechadv.2011.07.019] [PMID]
27. Soria-Guerra, R.E., Alpuche-Solís, A.G., Rosales-Mendoza, S., Moreno-Fierros, L., Bendik, E.M., Martínez-González, L. and Korban, S.S. (2009). Expression of a multi-epitope DPT fusion protein in transplastomic tobacco plants retains both antigenicity and immunogenicity of all three components of the functional oligomer. Planta, 229: 1293-1302. [DOI:10.1007/s00425-009-0918-2] [PMID] [PMCID]
28. Spök, A., Karner, S., Stein, A. and Rodríguez-Cerezo, E. (2008). Plant molecular farming: opportunities and challenges. JRC Scientific and Technical Reports. 28: 153-172. [DOI:10.1080/07388550802046624] [PMID]
29. Yarden, Y. (2001). Biology of HER2 and its importance in breast cancer. Oncology, 61: 1-13. [DOI:10.1159/000055396]
30. Zahnd, C., Kawe, M., Stumpp, M.T., de Pasquale, C., Tamaskovic, R., Nagy-Davidescu, G., Dreier, B., Schibli, R., Binz, H.K. and Waibel, R. (2010). Efficient tumor targeting with high-affinity designed ankyrin repeat proteins: effects of affinity and molecular size. Cancer Research, 70: 1595-1605. [DOI:10.1158/0008-5472.CAN-09-2724] [PMID]
31. Zahnd, C., Wyler, E., Schwenk, J.M., Steiner, D., Lawrence, M.C., McKern, N.M., Pecorari, F., Ward, C.W., Joos, T.O. and Plückthun, A. (2007). A designed ankyrin repeat protein evolved to picomolar affinity to Her2. Journal of Molecular Biology, 369: 1015-1028. [DOI:10.1016/j.jmb.2007.03.028] [PMID]
32. Zakerghoran, B., Rajabi-Memari, H., Nabati-Ahmadi, D. and Siahmard, M. (2014). Cloning, transformation and stable expression of a fusion of human interferon gamma and bar genes in tobacco plant (Nicotiana tobaccum cv. xanthi). Plant Genetic Researches, 1(1): 27-36 (In Persian). [DOI:10.29252/pgr.1.1.27]
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Ehsasatvatan M, Baghban Kohnehrouz B. Homoplasmic Stability and Cytoplasmic Inheritence of DARPin G3 Scaffold Protein in Generative and Vegetative Propagation of Transplastoic Tobacco Plants. pgr 2023; 9 (2) :1-14
URL: http://pgr.lu.ac.ir/article-1-262-en.html


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Volume 9, Issue 2 (2023) Back to browse issues page
پژوهش های ژنتیک گیاهی Plant Genetic Researches
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