Gene engineering of glycosylation system in rice cells for production of mammalian glycoproteins

• Main Authors: Wei-Cho Hsu, 徐暐焯
• Other Authors: 黃麗芬
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/45372121324257763869

碩士 === 元智大學 === 生物科技與工程研究所 === 98 === Plant cells are applied to produce many valuable recombinant proteins. Because of its low cost during culture and safety (plants rarely transmit human disease), plant cells are considered as a commercial potential hosts to express foreign proteins. However, application of recombinant mammalian glycoproteins in transgenic plants usually has problems in biological activity, protein stability and immunity, because the glycosylation system in plant cells is not compatible with that in mammalian cells. Therefore, a current challenge for the plant-based recombinant protein expression system is how to produce recombinant mammalian glycoproteins with original biological activities. Rice suspension cells have been applied to express several recombinant proteins. We tried to humanize N-glycosylation pathways in order to get the most benefits without the side effects of recombinant mammalian glycoproteins produced in transgenic rice cells. Two approaches were applied in this study to engineer the human-based N-glycosylation system in transgenic rice cell lines: one is to reduce (knock down) three endogenous plant specific glycosyltransferases (expressed ubiquitously throughout the rice plant) by RNA interference (RNAi) approach; the other is to ectopic express human β-1.4-glycosyltransferase. To repress the expression of plant specific β-1,2-xylosyltransferrase (XT), RNAi strategy was applied to make XT RNAi (XTi) transgenic rice cell lines. Based on RT-PCR analysis, 31 out of 36 transgenic lines showed the reduced expression level of XT. Total glycoprotein patterns were detected by immunostaining of rice protein extract with a horseradish peroxidase (a plant origin glycoprotein) antibody. Compared to non-transgenic wild-type plants, two XTi cell lines showed the reduced level of total glycoproteins, implying that plant specific xylose was decreased. The same approach was applied to two α-1,3- fucosyltransferases, FT1 and FT2, to knock down the specific FT isoforms. No obvious difference was found in their total glycoprotein patterns. In this study, human β-1,4-galactosyltransferase (hGT) cDNA was expressed in transgenic rice cells. A total of eight hGT putative transgenic lines all contained hGT mRNA. The hGT::GFP fusion protein was localized to the ER and Golgi, where glycosylation happens. Through engineering of N-glycosylation pathways in this study, we will know more about N-glycosylation and its physiological mechanism in rice cells. In addition, continued efforts will be made to improve rice expression system, and it will extend its advantages in pharmaceutical glycoprotein production.