Abstract
A reproducible method for cryopreservation of transgenic rice cells (Oryza sativa L. cv. Dongjin) producing recombinant human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) has been established. Here, we assessed recovery media and investigated recombinant protein homogeneity after long-term preservation. For recovery of cryopreserved transgenic rice cells, AA medium was suitable in terms of both morphology and production of hCTLA4Ig. There were no differences in cell growth, sugar consumption, and hCTLA4Ig production between non-cryopreserved and cryopreserved cells for up to 1 month. hCTLA4Ig produced from cryopreserved cells was identical that of hCTLA4Ig from non-cryopreserved cells, as determined by analysis of its molecular weight and isoforms. For long-term preservation, cell viability was stably maintained at 61% for 26 months. In conclusion, these results demonstrate the possibility for reproducible cryocell-banking of transgenic rice cells without changes in the characteristics of cells and target proteins.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Tekoah, Y., A. Shulman, T. Kizhner, and I. Ruderfer (2015) Large-scale production of pharmaceutical proteins in plant cell culture-the protalix experience. Plant Biotechnol. J. 13: 1199–1208.
Rosales-Mendoza, S. and M. A. Tello-Olea (2015) Carrot cells: A Pioneering platform for biopharmaceuticals production. Mol. Biotechnol. 57: 219–232.
Jung, J. -W., N. S. Kim, S. -H Jang, Y. -J. Shin, and M. -S. Yang (2016) Production and characterization of recombinant human acid a-glucosidase in transgenic rice cell suspension culture. J. Biotechnol. 226: 44–53.
Kuo, Y. -C., C. -C. Tan, J. -T. Ku, W. C. Hsu, S. -C. Su, C. -A. Lu, and L. -F. Huang (2013) Improving pharmaceutical protein production in Oryza sativa. Int. J. Mol. Sci. 14: 8719–8739.
Kwon, J. -Y., S. -H. Jeong, J. -W. Choi, Y. -Y. Pak, and D. -I. Kim (2013) Assessment of long-term cryopreservation for production of hCTLA4Ig in transgenic rice cell suspension cultures. Enz. Microb. Tech. 53: 216–222.
Whitmer, S., C. Canel, R. Van Der Heijden, and R. Verpoorte (2003) Long-term instability of alkaloid production by stably transformed cell lines of Catharanthus roseus. Plant Cell Tiss. Org. 74: 73–80.
James, E. and J. M. Lee (2006) Loss and recovery of protein productivity in genetically modified plant cell lines. Plant Cell Rep. 25: 723–727.
Boisson, A. -M., E. Gout, R. Bligny, and C. Rivasseau (2012) A simple and efficient method for the long-term preservation of plant cell suspension cultures. Plant Methods 8: 4.
Cho, J. -S., S. M. Hong, S. -Y. Joo, J. -S. Yoo, and D. -I. Kim (2007) Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig. Appl. Microbiol. Biot. 73: 1470–1476.
Jain, M., A. Nijhawan, A. K. Tyagi, and J. P. Khurana (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem. Biophys. Res. Commun. 345: 646–651.
Livak, K. J. and T. D. Schmittgen (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 24: 402–408.
Yu, D., M. -N. Song, J. -A. Lim, and D. -I. Kim (2008) Effects of culture media on hCTLA4Ig production and protein expression patterns in transgenic rice cell suspension cultures. Biotechnol. Bioproc. Eng. 13: 424–430.
Wesley-Smith J., C. Walters, P. Berjak, and N. W. Pammenter (2004) The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifoliata (L.). Cryolett. 25: 129–138.
Kaczmarczyk, A., N. Shvachko, Y. Lupysheva, M. -R. Hajirezaei, and E. R. J. Keller (2008) Influence of alternating temperature preculture on cyropreservation results for potato shoot tips. Plant Cell Rep. 27: 1551–1558.
Mustafa, N. R., W. De Winter, F. Van Iren, and R. Verpoorte (2011) Initiation, growth and cryopreservation of plant cell suspension cultures. Nat. Protoc. 6: 715–742.
Hayashi, T., C. Ohsumi, Y. Kato, H. Yamanouchi, K. Toriyama, and K. Hinata (1994) Effects of amino acid medium on cell aggregation in suspension-cultured rice cells. Biosci. Biotech. Biochem. 58: 256–260.
Rabilloud, T. (1996) Solubilization of proteins for electrophoretic analyses. Electrophoresis 17: 813–829.
Lüllau, E., A. Kanttinen, J. Hassel, M. Berg, A. Haag-Alvarsson, K. Cederbrant, B. Greenberg, C. Fenge, and F. Schweikart (2003) Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant β-secretase. Biotechnol. Prog. 19: 37–44.
Faye, L., A. Boulaflous, M. Benchabane, V. Gomord, and D. Michaud (2005) Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23: 1770–1778.
Gomord, V., P. Chamberlain, R. Jefferis, and L. Faye (2005) Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol. 23: 559–565.
Drakakaki, G. and A. Dandekar (2013) Protein secretion: How many secretory routes does a plant cell have? Plant Sci. 203–204: 74–78.
Zhu, L., Q. Guo, H. Guo, T. Liu, Y. Zheng, P. Gu, X. Chen, H. Wang, S. Hou, and Y. Guo (2014) Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry. MAbs. 6: 1474–1485.
Sethuraman, N. and T. A. Stadheim (2006) Challenges in therapeutic glycoprotein production. Curr. Opin. Biotechnol. 17: 341–346.
Kang, S. -H., H. -S. Jung, S. -J. Lee, C. -I. Park, S. -M. Lim, H. Park, B. -S. Kim, K. -H. Na, G. -J. Han, J. -W. Bae, H. -J. Park, K. -C. Bang, B. -T. Park, H. -S. Hwang, I. -S. Jung, J. -I. Kim, D. -B. Oh, D. -I. Kim, H. Yagi, K. Kato, D. -K. Kim, and H. -H. Kim (2015) Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspensioncultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig. Glycoconj. J. 32: 161–172.
Strasser, R. (2016) Plant protein glycosylation. Glycobiol. 26: 926–939.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kang, SH., Choi, HY., Cho, JS. et al. Assessment of Recovery Medium for Production of hCTLA4Ig after Cryopreservation in Transgenic Rice Cells. Biotechnol Bioproc E 23, 218–227 (2018). https://doi.org/10.1007/s12257-017-0477-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-017-0477-5