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1 Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006, pp 초파리 S2 세포시스템에서녹색형광단백질생산을위한재조합배큘로바이러스의감염조건들의영향 o Ç i Ç Çy q, q e q 31 ( o 21p r, o 23p }ˆ) Effects of Recombinant Baculovirus Infection Conditions on Production of Green Fluorescent Protein in Drosophila S2 Cells Hye Sook Cho, Yeon Kyu Kim, Kyoung Ro Kim and Hyung Joon Cha Division of Molecular and Life Sciences & Department of Chemical Engineering,GPohang University of Science and Technology, San 31, Hyoja-dong, Nam-gu, Pohang , Korea (Received 21 November 2005; accepted 23 December 2005) k o orq p dp ml p l Ž Drosophila melanogaster S2 l p p d/s2 edšp kr p dp qr Ž v k S2 p qrp r edšp. l l n r v p n v(green fluorescent protein) p eˆ qs p d pn l S2 lp ms l m l m. qs p dp S2 lp ms p multiplicity of infection(moi),, p d nkp re, p d ~ r vp ke p d r S2 ~p sq vl ˆn ke p r m. MOI p rp p p pp o l sp mp kp lv n p r e v p errp kl p MOI ov p n 100 mm krel p dm S2 r kp p MOI rrp p 30p, p d k e p 1.5 e p r p f p d t p S2l p pl., p d krel n p 2.4Íl q sp mp p d nkp 10 eˆp f p o ~ eˆ n vp pp pp f rp p dl p p tp pl. p l Ž kre l ˆ p p q ˆ ov m p p pl. p dp mp p l p m ke p 24e l p vp p ˆ l. h Abstract The baculovirus-insect Drosophila melanogaster S2 cell system combines advantages of conventional baculovirus system and non-lytic S2 cell system because baculoviruses can infect non-permissive cells such as mammalian and Drosophila S2 cells but cannot replicate themselves inside the cells. In the present work, we investigated effects of infection conditions on production of green fluorescent protein (GFP) as a target protein using this baculovirus-s2 cell system. Even though higher MOI and longer baculovirus contact time showed better GFP expression yield during the shorter period, overall protein yield could be lower during the longer period due to the relatively higher cell detachment and lysis (lower cell viability). In addition, maintaining high MOI will be not practical for large-scale cell culture. Therefore, instead of maintaining high MOI, we found that high initial cell number and concentrated (10X) baculovirus volume can confer comparable protein expression even under the moderate MOI condition. Also, we found that the postinfection time that is connected to state of cells after infection was an important factor for production yield. Key words: Baculovirus, Drosophila S2 Cells, Infection, Condition, Green Fluorescent Protein To whom correspondence should be addressed. hjcha@postech.ac.kr 40

2 Ž S2 edšl v p o qs p dp ms p m Ž Drosophila melanogaster Schneider line 2(S2) t d pn t p Ž ppˆv k krr p v r edšp p l m [1-3]. Ž p qrp p vr p 1,000 r v p p, p tp n orq p o l krrp t d v p l m e p v k rp p. o m p r l o vp lp p tp p ov p q sr rp q tn [3]. S2 edšp l v n p v o rq p prp l r p [4-6]. S2 edšp qr l, k o p n v p el krrp eˆ p p r p p r k. n orq v p p orqrp kr p p l p [7]. t p e vr p n p, p p neo( r G418l orq)m hph(hygromycin Bl orq) p rp ƒ n p ƒ p l rp l t l rv kp p p o [8]., r vp srp kr v r tl dv k [9]. p d Spodoptera frugiperdam Trichoplusia ni p p l p n v l pn l m [10-12]. qs p d p om p t meˆ p n vp p v p., e m r p n l tl p t n vp p, r vp v e p p [13, 14]. p p d edšp Ž l v v p r v p e mmp rm pl vr edšp l. o Ž l p d mp p d or~ l pp p d r r pl v k p orqp p r p lvv k [15, 16]. er p dp polyhedrin Ž l r p r v k p k r [15]. v, Rous os p dp r p d chloramphenicol acetyltransferase orq pl [15]., r p d Ž tl metallothionein p heat shock m p Ž l o l p p p d l p p p l [16, 17]. p r p qr p p d mp Ž v kk, rp r vp vr p lv., p d p tp n orq p S2 r pp p, r p mr ( e, n l m)l l r v p t p el l rp pl p [17]. np, r vp srp qs p d p m p eˆp f. l l qs d pn l Ž S2 l n vp o l vp ms p m l l s m. m o n vp Ž Aequorea victorial o v(green fluorescent protein; GFP)pl. p vp ee p k p l l vp s pq, p n lp q ~rp d p rl n p [11, 12] mo m (copper sulfate)l p p o metallothionein pn l vp S2 l eˆ p qs p d vmt-gfp[18] n mp p p dp titer pfu ml -1 pl h mo m i S2 p vmt-gfp qs p d m l vp p e m. kp 6-well plate, 60 mm kre 100 mm krel mp 10Í IMSm 300 µg ml -1 hygromycin B(Sigma) 4 ml M3 vl 1.6~ cells ml p v -1 ˆo. k v MOI e k 1~2,000p MOI v vmt-gfp p d nkp n v l ~ 27 Cl l t o 1~24e k k m. p d v l n v ~ m. m S2 vp o l s 500 µm o m. 2-3., s o hemacytometer(fisher Scientific) n l l, s trypan blue(sigma) 0.4Í r m l r m. vp kp q p rr qs v(clontech)p Western blotsp r tp k r m. vp v n l, k p 5,000 rpmp 10 oe l v p. v p SDS sample buffer(10í sodium dodecyl sulfate (SDS), 10Í β-mercaptanol, 0.3 M Tris-HCl (ph 6.8), 0.05Í bromophenol blue, 50Í glycerol) l, 5 k p 12.5Í SDS- k k p r r m (SDS-PAGE)p l m, pl r m rp Hybond TM -PVDF (Amersham Pharmacia))p m. 5Í ˆv o TBS buffer(20 mm Tris-HCl, 500 mm NaCl, ph 7.5)l 1e k pp tv, p p Š o p - v ~(1:1000 ; Santa Cruz Biotechnology) eq alkaline phosphatase m o p -Š p ~(1:1000 ; Sigma) ~ nk(1í ˆv o TTBS (TBS with 0.05Í Tween-20)l p nk)l l ml 1e k pe. TTBSm TBS l rp }p, o alkaline Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

01~50 p o p rp 3~10p ˆ MOIl qs vp sp p p. Ž S2 o p p d r p p MOI n k. p p l p d m eˆ p r n p m pp p p [20]. t, v, ( m e ), MOI s vp p v pl r p } l s p o p p [21].

3 42 s Ë l Ë Ë t phosphatasep v(fast red tr/naphthol as-mx tablet sets; Sigma)p ~ m v pp tv m. p dƒp, v vp l mp, Gel-Pro Analyzer ol (Media Cybernetics) m. 3. y 3-1. MOI s i i p d pn edšl MOI rkp t rp pqp. p d/ (Sf9 Hi-five) ed Šp vrp l rp 1 rp edšp l, p rp prp mp o rr MOI qs p d ~ rl p rp v q k. k l po p p m pl rrp m p l, p kk [19]. Sf-9 p r n l p rp p d MOI 0.01~50 p o p rp 3~10p ˆ MOIl qs vp sp p p. Ž S2 o p p d r p p MOI n k. p p l p d m eˆ p r n p m pp p p [20]. t, v, ( m e ), MOI s vp p v pl r p } l s p o p p [21]. l l n p MOI s m Western blot p l p p m (Fig. 1). 6-well platel MOI 2,000 v v eˆ v vp p v kk. p v kp Drosophila S2 l p p d pq p prp opp l eˆ vp p. p p o l l ; m 8e k, v p 800 v m [22]., p dp kp v e ˆ o p meˆ n β-galactosidase p l. Fig. 1. Effects of MOI on GFP production in vmt-gfp baculovirus/ S2 cells. Inner picture is GFP bands from Western blot analysis. Initial cell number was cells ml -1 in 6-well plate, baculovirus incubation time was 1.5 h, post-infection time was 24 h, and post-induction time was 42 h. o44 o k Fig. 2. Effects of MOI and initial cell number on GFP production in vmt-gfp baculovirus/s2 cells. Incubation time was 1.5 h, postinfection time was 24 h, and post-induction time was 42 h. Comparison of specific GFP expression for 60 mm dish culture MOIj { jm ƒ MOIl p l r vp p v eˆ p v n p kl n p p d n p er kl p ƒ r rp p. MOIm mp p k l rr p d ~ kp p ˆ. r v p p p v e 1.7~2 r p v p p p Western blot p l kk pl (Fig. 2). p Ž p k 60 mm 100 mm k rep l l ltl (data not shown). v, MOI p l r vp p p p. kp Fig. 1l k p p, v p m l p MOIl v kk. l, MOIm n v p tn p, r l MOIp v l e r k p. s l rr MOI s p s lk m kh z s i i r~ kre l p d v p r p r~ krep r p p l vm p d p pp. v, p pp l p p d kre l v l t p. p p p MOI p v p d p n l l n sp m p t p ltl (Fig. 3(a)). m s o p p d n p d n p k (Fig. 3(b)). p d p vrp Ž m edšp. Sf9 l p d, pp t p n l p. p p l p k p. kp l l 100 mm krep p

Ž S2 edšl v p o qs p dp ms p m 43 Fig. 4. The effect of MOI and incubation time with 10X concentrated vmt-gfp baculovirus on (a) specific GFP production and (b) cytotoxicity in wild type Drosophila S2 cells.

4 Ž S2 edšl v p o qs p dp ms p m 43 Fig. 4. The effect of MOI and incubation time with 10X concentrated vmt-gfp baculovirus on (a) specific GFP production and (b) cytotoxicity in wild type Drosophila S2 cells. Initial cell number was cells ml -1, post-infection was 24 h, and postinduction was 42 h. Fig. 3. Comparison of 10X concentrated and original vmt-gfp baculovirus stocks in wild type Drosophila S2 cells. (a) Specific GFP amount and (b) cytotoxicity. Incubation time was 1.5 h, post-infection time was 24 h, and post-induction time was 42 h for 100 mm dish culture. l r~ p d 2.4Íp nl rp p d m s p ll. 100 mm krel p 2.4Í p ml n p d n vl 1.2 ml p p p p mr re p. r~ p d 1.2 ml v qs p d n l p f l pl., p d n v ~ p f eˆ p p p d v p p tp p. vmt-gfp qs p dp n, p dp MOI 30p m s p ˆ mp vp 20Íp p m (Fig. 3) m h m s i i p d ke p p dm p r e p rp. e p n p d k e l sp r v pp k pl (data not shown). Fig. 4 k MOIl ke l v p p lt. p p d ke p v pp pp k plp (Fig. 4(a)) p p s k p ltl (Fig. 4(b)). p Ž k m Fig. 5. Effects of incubation time with 10X concentrated vmt-gfp baculovirus on (a) cell viability and (b) cytotoxicity. MOI was 30, initial cell number was cells ml -1, post-infection was 24 h, and post-induction was 42 h. p kr l r. Fig. 5 l l r m s p MOI 30l 1e vp k e k s 80Í p lt p. n pl pl o p, 1e p rp p d k e p r m. p d m p p opp p d mp o Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

44 s Ë l Ë Ë t y Fig. 6. Effect of incubation time after baculovirus infection on GFP expression in vmt-gfp/drosophila S2 cells. Western blot analysis was performed to detect GFP production.

5 44 s Ë l Ë Ë t y Fig. 6. Effect of incubation time after baculovirus infection on GFP expression in vmt-gfp/drosophila S2 cells. Western blot analysis was performed to detect GFP production. e p m l n p p v, rp l rp lp ppˆ e p [35] m i h m s i i m ke p rp p d m l ~p r ml l kr ˆp k p p. p d mp q rv ˆ v kk, p po m l n p p l p lp m (data not shown). np, p d r l qlrp r v k. m p m ke p p kr l l ppl, r l p p p dp r p pl rkp p. CMV-IE p sr l p v orq v m p l p rp m 24e l v p p [20]. 24e p rp m ke pp e p l k l (data not shown) p Western blot p l p pl (Fig. 6). 4. MOI p rp p p pp o l s p m. er kp l p dm S2 r kp tn p MOI 30p, p dmp k e p 1.5e p r p f p d S2 l p pl., p d nkp 10 eˆp f o ~ eˆ n vp pp pp f p dl p p tp pl. p dp m p p l p m ke p 24e l vp p q spp p m. l v q p l qvo l(krf-2004-d00181) l(brain Korea) 21p p p lp pl. o44 o k 1. Schneider, I., Cell Lines Derived from Late Embryonic Stages of Drosophila melanogaster, J. Embryol. Exp. Morphol., 27, (1972). 2. van der Straten, A., Johansen, H., Rosenberg, M. and Sweet, R. W., Introduction and Constitutive Expression of Gene Products in Cultured Drosophila Cells Using Hygromycin B Selection, Curr. Methods Mol. Cell Biol., 1, 1-8(1989). 3. Johansen, H., van der Straten, A., Sweet, R., Otto, E., Maroni, G. and Rosenberg, M., Regulated Expression at High Copy Number Allows Production of a Growth-inhibitory Oncogene Product in Drosophila Schneider Cells, Genes Div., 3, (1989). 4. Kirkpatrick, R. B., Ganguly, S., Angelichio, M., Griego, S., Shatzman, A., Silverman, C. and Rosenberg, M., Heavy Chain Dimers as well as Complete Antibodies are Efficiently Formed and Secreted from Drosophila via a BiP-mediated Pathway, J Biol Chem., 270, (1995). 5. Li, B., Tsing, S., Kosaka, A. H., Nguyen, B., Osen, E. G., Bach, C., Chan, H. and Barnett, J., Expression of Human Dopamine Beta-hydroxylase in Drosophila Schneider 2 Cells, Biochem J., 313, 57-64(1996). 6. Lehr, R. V., Elefante, L. C., Kikly, K. K., O Brien, S. P. and Kirkpatrick, R. B., A Modified Metal-ion Affinity Chromatography Procedure for the Purification of Histidine-tagged Recombinant Proteins Expressed in Drosophila S2 Cells, Protein Expr Purif., 19, (2000). 7. Corchero, J. L. and Villaverde, A., Plasmid Maintenance in Escherichia coli Recombinant Cultures is Dramatically, Steadily, and Specifically Influenced by Features of the Encoded Proteins, Biotechnol Bioeng., 58, (1998). 8. Dobrosotskaya, I. Y., Goldstein, J. L., Brown, M. A. and Rawson, R. B., Reconstitution of Sterol-regulated Endoplasmic Reticulum-to-Golgi Transport of SREBP-2 in Insect Cells by Co-expression of Mammalian SCAP and Insigs, J Biol Chem., 278, (2003). 9. Liu, X., Constantinescu, S. N., Sun, Y., Bogan, J. S., Hirsch, D., Weinberg, R. A. and Lodish, H. F., Generation of Mammalian Cells Stably Expressing Multiple Genes at Predetermined Levels, Anal Biochem., 280, 20-28(2000). 10. Miller, M. K., Baculoviruses as Gene Expression Vectors, Ann Rev Microbiol., 42, (1988). 11. Cha, H. J., Pham, M. Q., Rao, G. and Bentley, W. E., Expression of Green Fluorescent Protein in Insect Larvae and its Application for Foreign Protein Production, Biotechnol Bioeng., 56, (1997). 12. Cha, H. J., Dalal, N. G., Vakharia, V. N. and Bentley, W. E., Expression and Purification of Human Interleukin-2 Simplified as a Fusion with Green Fluorescent Protein in Suspended Insect Sf-9 Cells, J Biotechnol., 69, 9-17(1999). 13. O Reilly, D. R. and Miller, L. K., Expression and Complex Formation of Simian Virus 40 Large T Antigen and Mouse p53 in Insect Cells, J Virol., 62, (1988). 14. Hu, Y. C. and Bentley, W. E., Effect of MOI Ratio on the Composition and Yield of Chimeric Infectious Bursal Disease Virus-like Particles by Baculovirus Co-infection: Deterministic Predictions and Experimental Results, Biotechnol Bioeng., 75, (2001).

6 Ž S2 edšl v p o qs p dp ms p m Carbonell, L. F., Klowden, M. J. and Miller, L. K., Baculoviursmediated Expression of Bacterial Genes in Dipteran and Mammalian Cells, J Virol., 56, (1985). 16. Morris, T. D. and Miller, L. K., Promoter Influence on Baculovirus-mediated Gene Expression in Permissive and Nonpermissive Insect Cell Lines, J Virol., 66, (1992). 17. Lee, D. F., Chen, C. C., Hsu, T. A. and Juang, J. L., A Baculovirus Superinfection System: Efficient Vehicle for Gene Transfer into Drosophila S2 Cells, J Virol., 74, (2000). 18. Cho, H. S., Kim, Y. K. and Cha, H. J., Expression of Double Foreign Protein Types Following Recombinant Baculovirus Infection of Stably Transfected Drosophila S2 Cells, Enzyme and Microbial Technology., 35, (2004). 19. Joseph, M., Gene Expression System, Academic press., (1999). 20. Hu, Y. C., Tsai, C. T., Chang, Y. J. and Huang, J.U., Enhancement and Prolongation of Baculovirus-mediated Expression in Mammalian Cells: Focuses on Strategic Infection and Feeding, Biotechnol. Prog., 19, (2003). 21. Friedrich, A., Erika, S., Iain, B. H. W. and Leopold, M., Insect Cells as Hosts for the Expression of Recombinant Glycoproteins, Glycoconjugate jornal., 16, (1999). 22. Duisit, S., Saleun, S., Douthe, S., Barsoum, J., Chadeuf, G. and Moullier, P., Baculovirus Vetor Requires Electrostatic Interactions Including Heparin Sulfate for Efficient Gene Transfer in Mammalian Cells, The J. of gene medicine., 1, (1999). Korean Chem. Eng. Res., Vol. 44, No. 1, February, 2006

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<30372E31362D323028BDC5C7F6C5C32DB9CCB1B970626D292E666D> k m v r1 r1 16~20 (2009) J. Korean Academy of Managed Care Pharmacy Vol. 1, No. 1 Pharmacy Benefit Management iç 1# x sƒ, 1 w w w Overview on Pharmacy Benefit Management in USA Kyung Eob ChoiGand Hyun