[ 생물 ] 과학고 R&E 결과보고서 광우병과바이러스오염으로부터안전한 엔테로키나아제생산 연구기간 : 2013. 3. 1 ~ 2014. 2. 28 연구책임자 : 이균오 ( 경상대생화학전공 ) 지도교사 : 이팔홍 ( 경남과학고생명과학과 ) 참여학생 : 정승현 ( 경남과학고 1학년 ) 강한울 ( 경남과학고 1학년 ) 김면환 ( 경남과학고 1학년 ) 임호진 ( 경남과학고 1학년 )
(PrPSc), EK (glycan). EK. mrna (enterokinase: EK) construct, mrna EK - - mrna - Oligonucleotide primer RT-PCR cdna - TA EK cdna construct - EK binary vector, - EK Ti - (floral dipping method) EK T-DNA - T1 - T-DNA copy : - copy T-DNA (homozygote) - (immunoblotting) EK - Tandem affinity purification(tap) EK - GST-GFP - EK (enterokinase), (PrPSc), (viral contamination), (Plant made pharmaceuticals), (glycosylation)
1 1. (EK). EK (serine protease) (fusion proteins) (affinity tag). (PrPSc), EK (glycan). ( ) EK. 2.. (Human Genome Project)., 2007 11 Knome ( : Personal Genome).. 2.,,,,,. (molecule farming). (post-translational modification),
. (enterokinase) (precursor) (trypsinogen) (trypsin) (serine protease) (Kunitz, 1939; Light and Janska, 1989). 35 kda (light chain) 115 kda (heavy chain) (disulfide bond) (Yuan and Hua, 2002). Asp-Asp-Asp-Asp-Lys (lysine) (fused protein) - (Light and Fonseca, 1984). ph(4.5-9.5) (4-45 ) (Yuan and Hua, 2002). (affinity tag).. (E. coli).. (Edward. R, 1993). thioredoxin (Trx) (Yuan and Hua, 2001). (Tan and Wang and Zhao, 2007).. (endotoxin) (Huang, McDonald, 2009)..
... (Korean bovine) (duodenum) (Arabidopsis thalina)...,,,. (Yuan and Hua, 2002; Tan et al., 2007; Chun et al., 2011). Chinese hamster ovary (CHO) (human fibroblast) (Schmidt et al., 1997; Kariya et al., 2002; Jones et al., 2003). (PrPSc)..,, (Wildt and Gerngross, 2005; Cox et al., 2006; Shaaltiel et al., 2007)...,,
(Pujol et al., 2005; Lienard et al., 2007; He et al., 2012)..
2 (1) EK mrna ( ) ( ) mrna ( ) Primer (RT-PCR) cdna ( ) TA EK cdna ( ) EK binary vector (1), ( ) EK Ti ( ) (floral dipping method) EK T-DNA ( ) T1 ( ) T-DNA copy : ( ) copy T-DNA (homozygote) (2) ( ) (immunoblotting) ( ) GST-Asp-Asp-Asp-Asp-Lys-GFP ( ) GST-Asp-Asp-Asp-Asp-Lys-GFP ( )
3 1. mrna, cdna (Enterokinase: EK) (serine protease). mrna, (RT-PCR) cdna. cdna primer 700 bp cdna ( 1A). cdna TA pgem T-easy Vector. NCBI Bos taurus 596 (Guanine; G) 669 (cytosine; C) (Adenine; A) (Guanine; G), 219 (valine; Val) (methionine; Met) ( 1B). cdna binary vector pgreenii 0179 ( 1C).
1. cdna (A) (B) binary vector (C). 2. ( ),.,,. GV3101(+pSoup) (electroporation). (floral dipping method) T-DNA (Clough and Bent, 1998). hygromycin MS. 12 T1, T1 Streptavidin Binding Peptide (SBP) SBP-tag
6 ( 2). 3:1( : ) single-copy T-DNA 5 T2. 2. 3.. GST-Asp-Asp-Asp-Asp-Lys-GFP (Asp-Asp-Asp-Asp-Lys) Lys., -Asp-Asp-Asp-Asp-Lys- ( 3A). GST, GFP Asp-Asp-Asp-Asp-Lys. (BL21). 60 kda ( 3B). glutathione (GSH) agarose bead affinity chromatography.
3.. EV:, ES:,. (GST-Asp-Asp-Asp-Asp-Lys-GFP). 20 μg 10 μg 37 1, 2, 4 ( 4). SDS-PAGE 4. ( 60 kda), 27 kda..
4.. ES:,, 4. N-. N- N-,. - oligosaccharyltransferase (OST) (Zufferey et al., 1995). NetNGlyc 1.0 Server 3 N- ( 5). N-. N-. N-,.
5. N-. N- N-. PNGase F Endo H. EK ( 6)., PNGase F Endo H SDS-PAGE. N-..
6. N-. C: EK, 1: (rek), 2: rek+pngase F, 3: rek+endo H.
4 (1) ( ) ( ) (1) ( ),,, ( ) ( ) ( )
5. mrna cdna, primer 700 bp cdna. 596 (Guanine; G) 669 (cytosine; C) (Adenine; A) (Guanine; G), 219 (valine; Val) (methionine; Met). cdna binary vector pgreenii0179.,. Streptavidin Binding Peptide (SBP) SBP-tag, GST-GFP. 3,.,..
6 Chun, H., Joo, K., Lee, J., and Shin, H.C. (2011). Design and efficient production of bovine enterokinase light chain with higher specificity in E. coli. Biotechnol Lett 33, 1227-1232. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Cox, K.M., Sterling, J.D., Regan, J.T., Gasdaska, J.R., Frantz, K.K., Peele, C.G., Black, A., Passmore, D., Moldovan-Loomis, C., Srinivasan, M., Cuison, S., Cardarelli, P.M., and Dickey, L.F. (2006). Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24, 1591-1597. He, X., Galpin, J.D., Tropak, M.B., Mahuran, D., Haselhorst, T., von Itzstein, M., Kolarich, D., Packer, N.H., Miao, Y., Jiang, L., Grabowski, G.A., Clarke, L.A., and Kermode, A.R. (2012). Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants. Glycobiology 22, 492-503. Jones, D., Kroos, N., Anema, R., van Montfort, B., Vooys, A., van der Kraats, S., van der Helm, E., Smits, S., Schouten, J., Brouwer, K., Lagerwerf, F., van Berkel, P., Opstelten, D.J., Logtenberg, T., and Bout, A. (2003). High-level expression of recombinant IgG in the human cell line per.c6. Biotechnol Prog 19, 163-168. Kariya, Y., Ishida, K., Tsubota, Y., Nakashima, Y., Hirosaki, T., Ogawa, T., and Miyazaki, K. (2002). Efficient expression system of human recombinant laminin-5. J Biochem 132, 607-612. Kunitz, M. (1939). Formation of Trypsin from Crystalline Trypsinogen by Means of Enterokinase. J Gen Physiol 22, 429-446. Lienard, D., Sourrouille, C., Gomord, V., and Faye, L. (2007). Pharming and transgenic plants. Biotechnol Annu Rev 13, 115-147. Light, A., and Fonseca, P. (1984). The preparation and properties of the catalytic subunit of bovine enterokinase. J Biol Chem 259, 13195-13198. Light, A., and Janska, H. (1989). Enterokinase (enteropeptidase): comparative aspects. Trends Biochem Sci 14, 110-112. Pujol, M., Ramirez, N.I., Ayala, M., Gavilondo, J.V., Valdes, R., Rodriguez, M., Brito, J., Padilla, S., Gomez, L., Reyes, B., Peral, R., Perez, M., Marcelo, J.L., Mila, L., Sanchez, R.F., Paez, R., Cremata, J.A., Enriquez, G., Mendoza, O., Ortega, M., and Borroto, C. (2005). An integral approach towards a practical
application for a plant-made monoclonal antibody in vaccine purification. Vaccine 23, 1833-1837. Schmidt, H.H., Genschel, J., Haas, R., Buttner, C., and Manns, M.P. (1997). Expression and purification of recombinant human apolipoprotein A-I in Chinese hamster ovary cells. Protein Expr Purif 10, 226-236. Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., Dym, O., Boldin-Adamsky, S.A., Silman, I., Sussman, J.L., Futerman, A.H., and Aviezer, D. (2007). Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J 5, 579-590. Tan, H., Wang, J., and Zhao, Z.K. (2007). Purification and refolding optimization of recombinant bovine enterokinase light chain overexpressed in Escherichia coli. Protein Expr Purif 56, 40-47. Wildt, S., and Gerngross, T.U. (2005). The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3, 119-128. Yuan, L.D., and Hua, Z.C. (2002). Expression, purification, and characterization of a biologically active bovine enterokinase catalytic subunit in Escherichia coli. Protein Expr Purif 25, 300-304. Zufferey, R., Knauer, R., Burda, P., Stagljar, I., te Heesen, S., Lehle, L., and Aebi, M. (1995). STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo. EMBO J 14, 4949-4960.
7 ( ) Enterokinase ( 1, jsh46789@daum.net) ( 1, qwere9708@naver.com) ( 1, kimmh1242@naver.com) ( 1, slyapple@naver.com) (, mon2082@hanmail.net) (, leeko@gnu.ac.kr) Ⅰ. 1. recombinant protein. recombinant protein. virus 2.,,,. (molecule farming) (post-translational modification), virus. (Enterokinase)
(precursor) (trypsinogen) (trypsin) (serine protease) (Kunitz, 1939; Light and Janska, 1989). Enterokinase 35 kda (light chain) 115 kda (heavy chain) (disulfide bond) (Yuan and Hua, 2002). Asp-Asp-Asp-Asp-Lys (lysine) C- (fused protein) N- (Light and Fonseca, 1984). ph(4.5-9.5) (4-45 ) (Yuan and Hua, 2002). Enterokinase affinity tag. Enterokinase Enterokinase. Enterokinase (E. coli) Enterokinase. Enterokinase. Enterokinase (Edward. R, 1993). Enterokinase Thioredoxin (Trx) (Yuan and Hua, 2001). Enterokinase, Enterokinase (Tan and Wang and Zhao, 2007). Enterokinase. (endotoxin). Enterokinase.. Enterokinase.
2. Enterokinase(EK) (serine protease) (fusion proteins) (affinity tag). Enterokinase, Enterokinase (glycan). ( ) Enterokinase. Ⅱ. 1. mrna, cdna Enterokinase gene cloning Enterokinase mrna.. mrna mrna (RT-PCR). RT-PCR cdna Enterokinase Primer Enterokinase. TA, Enterokinase vector cloning cloning cdna. Enterokinase binary vector vector. 2. ( ) Enterokinase Enterokinase Ti. seed (floral dipping method) Enterokinase T-DNA seed. seed
T1 copy T-DNA T1 (homozygote) Enterokinase seed. 3. Enterokinase (immunoblotting) Enterokinase. seed GST-Asp-Asp-Asp-Asp-Lys-GFP. GST-Asp-Asp-Asp-Asp-Lys-GFP seed Enterokinase. Enterokinase. Ⅲ. 1. 1. mrna, cdna Enterokinase gene cloning Enterokinase Enterokinase mrna, template (RT-PCR) cdna. cdna Enterokinase primer 700 bp Enterokinase cdna. ( 1A). Enterokinase cdna TA pgem T-easy Vector. NCBI Bos taurus Enterokinase light chain 596 (Guanine; G) 669 (cytosine; C) (Adenine; A) (Guanine; G), 219 (valine; Val) (methionine; Met) ( 1B). Enterokinase cdna genome recombinant binary vector pgreenii 0179 vector ( 1C).
1. Enterokinase cdna (A) Enterokinase (B) binary vector (C). 2. ( ) Enterokinase recombinant protein folding,., Enterokinase, Enterokinase. Enterokinase GV3101(+pSoup) (electroporation). (floral dipping method) Enterokinase T-DNA (Clough and Bent, 1998), hygromycin MS. 12 T1, T1
Streptavidin Binding Peptide (SBP) SBP-tag Enterokinase 6 ( 2). 3:1( : ) single-copy T-DNA 5 T2. 2. Enterokinase 3. Enterokinase. Enterokinase GST-Asp-Asp-Asp-Asp-Lys-GFP Enterokinase (Asp-Asp-Asp-Asp- Lys) Lys. Enterokinase, -Asp-Asp-Asp-Asp-Lys- ( 3A). GST, Enterokinase GFP Enterokinase Asp-Asp-Asp-Asp-Lys. (BL21). 60 kda ( 3B). glutathione (GSH) agarose bead affinity chromatography.
3. Enterokinase. EV:, ES:,. Enterokinase Enterokinase (GST-Asp-Asp-Asp-Asp-Lys-GFP) Enterokinase. 20 μg 10 μg 37 1, 2, 4 ( 4). SDS-PAGE 4. Enterokinase ( 60 kda), 27 kda. Enterokinase.
4. Enterokinase. ES:,, 4. Enterokinase N-. Enterokinase N- N-,. - oligosaccharyltransferase (OST) (Zufferey et al., 1995). Enterokinase NetNGlyc 1.0 Server 3 N- ( 5). N- Enterokinase. Enterokinase N-. N-, Enterokinase Enterokinase.
5. Enterokinase N-. Enterokinase N- Enterokinase N-. PNGase F Endo H. Enterokinase Enterokinase ( 6)., Enterokinase PNGase F Endo H Enterokinase SDS-PAGE. Enterokinase N-. Enterokinase Enterokinase.
6. Enterokinase N-. C: Enterokinase, 1: Enterokinase(rEK), 2: rek+pngase F, 3: rek+endo H. Ⅳ.,. mrna cdna, primer 700 bp cdna. 596 669, 219., Enterokinase. cdna binary vector pgreenii0179.,.
seed Streptavidin Binding Peptide (SBP) SBP-tag, GST-GFP. 3,.,.,. Ⅴ. Chun, H., Joo, K., Lee, J., and Shin, H.C. (2011). Design and efficient production of bovine Enterokinase light chain with higher specificity in E. coli. Biotechnol Lett 33, 1227-1232. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Cox, K.M., Sterling, J.D., Regan, J.T., Gasdaska, J.R., Frantz, K.K., Peele, C.G., Black, A., Passmore, D., Moldovan-Loomis, C., Srinivasan, M., Cuison, S., Cardarelli, P.M., and Dickey, L.F. (2006). Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24, 1591-1597. He, X., Galpin, J.D., Tropak, M.B., Mahuran, D., Haselhorst, T., von Itzstein, M., Kolarich, D., Packer, N.H., Miao, Y., Jiang, L., Grabowski, G.A., Clarke, L.A., and Kermode, A.R. (2012). Production of active human glucocerebrosidase in seeds of Arabidopsis thaliana complex-glycan-deficient (cgl) plants. Glycobiology 22, 492-503. Jones, D., Kroos, N., Anema, R., van Montfort, B., Vooys, A., van der Kraats, S., van der Helm, E., Smits, S., Schouten, J., Brouwer, K., Lagerwerf, F., van Berkel, P., Opstelten, D.J., Logtenberg, T., and Bout, A. (2003). High-level expression of recombinant IgG in the human cell line per.c6. Biotechnol Prog 19, 163-168.
Kariya, Y., Ishida, K., Tsubota, Y., Nakashima, Y., Hirosaki, T., Ogawa, T., and Miyazaki, K. (2002). Efficient expression system of human recombinant laminin-5. J Biochem 132, 607-612. Kunitz, M. (1939). Formation of Trypsin from Crystalline Trypsinogen by Means of enterokinase. J Gen Physiol 22, 429-446. Lienard, D., Sourrouille, C., Gomord, V., and Faye, L. (2007). Pharming and transgenic plants. Biotechnol Annu Rev 13, 115-147. Light, A., and Fonseca, P. (1984). The preparation and properties of the catalytic subunit of bovine Enterokinase. J Biol Chem 259, 13195-13198. Light, A., and Janska, H. (1989). Enterokinase (enteropeptidase): comparative aspects. Trends Biochem Sci 14, 110-112. Pujol, M., Ramirez, N.I., Ayala, M., Gavilondo, J.V., Valdes, R., Rodriguez, M., Brito, J., Padilla, S., Gomez, L., Reyes, B., Peral, R., Perez, M., Marcelo, J.L., Mila, L., Sanchez, R.F., Paez, R., Cremata, J.A., Enriquez, G., Mendoza, O., Ortega, M., and Borroto, C. (2005). An integral approach towards a practical application for a plant-made monoclonal antibody in vaccine purification. Vaccine 23, 1833-1837. Schmidt, H.H., Genschel, J., Haas, R., Buttner, C., and Manns, M.P. (1997). Expression and purification of recombinant human apolipoprotein A-I in Chinese hamster ovary cells. Protein Expr Purif 10, 226-236. Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., Dym, O., Boldin-Adamsky, S.A., Silman, I., Sussman, J.L., Futerman, A.H., and Aviezer, D. (2007). Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J 5, 579-590. Tan, H., Wang, J., and Zhao, Z.K. (2007). Purification and refolding optimization of recombinant bovine Enterokinase light chain overexpressed in Escherichia coli. Protein Expr Purif 56, 40-47. Wildt, S., and Gerngross, T.U. (2005). The humanization of N-glycosylation pathways in yeast. Nat Rev Microbiol 3, 119-128. Yuan, L.D., and Hua, Z.C. (2002). Expression, purification, and characterization of a biologically active bovine Enterokinase catalytic subunit in Escherichia coli. Protein Expr Purif 25, 300-304. Zufferey, R., Knauer, R., Burda, P., Stagljar, I., te Heesen, S., Lehle, L., and Aebi, M. (1995). STT3, a highly conserved protein required for yeast oligosaccharyl transferase activity in vivo. EMBO J 14, 4949-4960.