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Application of perfusion system for the production of hCTLA4Ig in transgenic rice cell cultures
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S4–S20 AN-P16 A renal glomerular cell culture model using a collagen vitrigel membrane and its application to the evaluation of serum filtration Maya Fukuda,1,2 Pi Chao Wang,1 and Toshiaki Takezawa2 Graduate School of Life and Environmental Sciences, University of Tsukuba, Tukuba, Japan 1 and National Institute of Agrobiological Sciences, Tukuba, Japan 2 Although the function of renal glomeruli has been known to filter the circulating blood in our body, its role is not only limited to provide a filtrating membrane but also to regulate the blood passing through basement membrane by glomerular cells. However, especially how the glomerular cells regulate the blood filtration remains unclear because no evaluation system has been established yet. We tried to elucidate the cellular regulation function and mechanism on blood filtration by using vitrigel a novel vitrigel membrane useful to both in vitro and in vivo system. Porcine glomeruli isolated from kidney were outgrown on the dish. Single culture system of epithelial cell and mesangial cells were conducted by seeding each cell at one side of vitrigel, respectively. Coculture system was performed by seeding one kind of cells at one side of vitrigel and then the other kind of cells at the opposite side of vitrigel. Culture of vitrigel without seeding any cell is used as control. The evaluation of filtration is conducted by adding serum at the top of vitrigel at control, single culture and co-culture system and the passing through serum at time intervals was collected and subject to SDS-PAGE following with silver stain. The amount change and molecular size of the proteins in the serum before and after filtrating through vitrigel were compared and evaluated. In the addition, the cellular morphology changes on vitrigel were also observed by microscopy. Single culture system of epithelial cells on vitrigel significantly regulated the serum filtration while that of mesangial cells showed no regulated function as compared to control. Microscopic observation showed that epithelial cells adhered to each other while mesangial cells distributed randomly in the dish culture. These facts imply that cell–cell interaction between epithelial cells may play an important role in regulating blood filtration in vivo. doi:10.1016/j.jbiosc.2009.08.046
AN-P17 Application of perfusion system for the production of hCTLA4Ig in transgenic rice cell cultures Yong-Suk Yang, Jun-Young Kwon, Su-Hwan Cheon, Sung-Hun Choi, Boreum Yun, Hye-Ran Lee, Ji-Yeon Han, Mi-Hee Yoo, Ji-Won Choi, and Dong-Il Kim Department of Biological Engineering, Inha University, 253 Yonghyun-dong, Nam-gu, Incheon, Republic of Korea Transgenic plant cell cultures have been considered as an attractive alternative system to produce recombinant proteins for industrial and pharmaceutical uses (1). Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was expressed in genet-
S13
ically modified rice cell suspension cultures using RAmy3D promoter. In this expression system, secretion of hCTLA4Ig into culture medium was performed with depletion of sugar although extensive cell growth was attainable with sugar-rich medium. Thus, cell lysis and degradation of target protein occur during the production phase, which limit the production efficiency (2). For that reason, supplementation of alternative energy source for the continuous production system is needed to enhance cell viability as well as the production level during the production period. In this study, the hCTLA4Ig production by the induction medium containing 8 mM glucose in 100mL flask was increased 1.3-fold compared to the control culture without glucose. In addition, the low glucose concentration in the induction medium could induce the cells to produce hCTLA4Ig and maintain the basal metabolism in a 5-L stirred tank bioreactor (STR) by perfusion culture system. The mean residence time of the medium in the 5-L STR was short enough to prevent the target protein from possible degradation. References 1. Kusanadi, A.R., Nikolov, Z.L., and Howard, A.J..: Production of recombinant proteins in transgenic plants: practical consideration, Biotechnol. Bioeng., 56, 473-484 (1997). 2. Terashima, M., Ejiri, Y., Hashikawa, N., and Yoshida, H.: Effects of osmotic pressure on human alpha1-antitrypsin production by plant cell culture, Biochem. Eng. J., 4, 31-36 (1999).
doi:10.1016/j.jbiosc.2009.08.047
AN-P18 Expression of recombinant mAb CO17-1A in tomato plants Ki-Hyun Yoo,1 Ji-Hye Lee,1 Kyung-Il Kim,1 Ha-Young Chung,1 Ho-Yong Chung,1 Ki-Sung Ko,2 and In-Sik Chung1 Graduate School of Biotechnology, Kyung Hee University, Yongin, Republic of Korea 1 and Department of Lif Science, WonKwang University, Iksan, Republic of Korea 2 Beet curly top virus (BCTV) belongs to geminivirus subgroup II. This virus infects a wide range of dicotyledonous plant hosts and has a monopartite genome. The mAb CO17-1A (IgG2a) recognizes the tumor-associated antigen GA733, which is highly expressed on human colorectal carcinomas (1). This mAb has proven to be efficacious in treating micrometastases and in preventing the recurrence of colorectal cancer in high-risk patients (2). mAb CO171A was the first murine anticarcinoma antibody tested in humans and has a well-documented safety record. However, mAb production using hybridoma technology is limited by the high cost associated with obtaining sufficient quantities and by the potential presence of animal pathogens (3). In this study, we examined the use of BCTV elements to enhance expression of recombinant mAb CO17-1A in tomato plants. Southern hybridization analysis showed that unit-length DNAs of replicated BCTV could be detected 3 and 6 days after the cultivation of Agrobacterium-inoculated leaf-disks of tomato plants. We are currently investigating the expression of mAb CO17-1A by RT-PCR and Western blot analysis in plants. In addition, the potential related to BCTV expression system for the production of cancer-preventive therapeutics will be discussed. This research is supported by a grant from Biogreen 21 Project (2007040103426).
AN-P17 Application of perfusion system for the production of hCTLA4Ig in transgenic rice cell cultures Yong-Suk Yang, Jun-Young Kwon, Su-Hwan Cheon, Sung-Hun Choi, Boreum Yun, Hye-Ran Lee, Ji-Yeon Han, Mi-Hee Yoo, Ji-Won Choi, and Dong-Il Kim Department of Biological Engineering, Inha University, 253 Yonghyun-dong, Nam-gu, Incheon, Republic of Korea Transgenic plant cell cultures have been considered as an attractive alternative system to produce recombinant proteins for industrial and pharmaceutical uses (1). Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was expressed in genet-
S13
ically modified rice cell suspension cultures using RAmy3D promoter. In this expression system, secretion of hCTLA4Ig into culture medium was performed with depletion of sugar although extensive cell growth was attainable with sugar-rich medium. Thus, cell lysis and degradation of target protein occur during the production phase, which limit the production efficiency (2). For that reason, supplementation of alternative energy source for the continuous production system is needed to enhance cell viability as well as the production level during the production period. In this study, the hCTLA4Ig production by the induction medium containing 8 mM glucose in 100mL flask was increased 1.3-fold compared to the control culture without glucose. In addition, the low glucose concentration in the induction medium could induce the cells to produce hCTLA4Ig and maintain the basal metabolism in a 5-L stirred tank bioreactor (STR) by perfusion culture system. The mean residence time of the medium in the 5-L STR was short enough to prevent the target protein from possible degradation. References 1. Kusanadi, A.R., Nikolov, Z.L., and Howard, A.J..: Production of recombinant proteins in transgenic plants: practical consideration, Biotechnol. Bioeng., 56, 473-484 (1997). 2. Terashima, M., Ejiri, Y., Hashikawa, N., and Yoshida, H.: Effects of osmotic pressure on human alpha1-antitrypsin production by plant cell culture, Biochem. Eng. J., 4, 31-36 (1999).
doi:10.1016/j.jbiosc.2009.08.047
AN-P18 Expression of recombinant mAb CO17-1A in tomato plants Ki-Hyun Yoo,1 Ji-Hye Lee,1 Kyung-Il Kim,1 Ha-Young Chung,1 Ho-Yong Chung,1 Ki-Sung Ko,2 and In-Sik Chung1 Graduate School of Biotechnology, Kyung Hee University, Yongin, Republic of Korea 1 and Department of Lif Science, WonKwang University, Iksan, Republic of Korea 2 Beet curly top virus (BCTV) belongs to geminivirus subgroup II. This virus infects a wide range of dicotyledonous plant hosts and has a monopartite genome. The mAb CO17-1A (IgG2a) recognizes the tumor-associated antigen GA733, which is highly expressed on human colorectal carcinomas (1). This mAb has proven to be efficacious in treating micrometastases and in preventing the recurrence of colorectal cancer in high-risk patients (2). mAb CO171A was the first murine anticarcinoma antibody tested in humans and has a well-documented safety record. However, mAb production using hybridoma technology is limited by the high cost associated with obtaining sufficient quantities and by the potential presence of animal pathogens (3). In this study, we examined the use of BCTV elements to enhance expression of recombinant mAb CO17-1A in tomato plants. Southern hybridization analysis showed that unit-length DNAs of replicated BCTV could be detected 3 and 6 days after the cultivation of Agrobacterium-inoculated leaf-disks of tomato plants. We are currently investigating the expression of mAb CO17-1A by RT-PCR and Western blot analysis in plants. In addition, the potential related to BCTV expression system for the production of cancer-preventive therapeutics will be discussed. This research is supported by a grant from Biogreen 21 Project (2007040103426).