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The effect of cell cycle on GFPuv gene expression in the baculovirus expression system
Journal of Biotechnology 93 (2002) 121– 129 www.elsevier.com/locate/jbiotec
The effect of cell cycle on GFPuv gene expression in the baculovirus expression system Takehiko Saito a, Takashi Dojima b, Masaru Toriyama a, Enoch Y. Park a,* a
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka Uni6ersity, 836 Ohya, Shizuoka 422 -8529, Japan b Production Technology Laboratory, Kaken Pharmaceutical Co. Ltd., 301 Gensuke, Fujieda 426 -8646, Japan Received 26 January 2001; received in revised form 6 August 2001; accepted 20 August 2001
Abstract The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G2/M phase with 4n DNA. In the case of G1 or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G2/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G1 phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G2/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G1 cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G1 or S phase-infection was 1.5–1.8-fold higher than that at G2/M phase-infection. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cell cycle; Sf9 cell; Baculovirus-insect cell expression system; GFPuv
1. Introduction The baculovirus expression system (BES) is widely used to express prokaryotic and eucaryotic proteins, because of its ease of use, high expression level (Luckow and Summers, 1988) and possibility of post-translational modifications, e.g. glucosylation and phosphorylation (Luckow, 1993). Many kinds of research into ways to improve the production of recombinant proteins * Corresponding author. Tel./fax: + 81-54-238-4887. E-mail address: [email protected] (E.Y. Park).
have been reported, including multiplicity of infection (MOI) (Licari and Bailey, 1991), cell line selection (Hink et al., 1991), culture condition (Jesionowski and Ataai, 1997; Taticek and Shuler, 1997; Wang et al., 1992) and an investigation of the infection process (Dee and Shuler, 1997). In mammalian cells, recombinant protein production and gene transfer using the virus were affected by host cell cycles. b-Galactosidase production rate in the S phase was higher than that in the G1 or G2/M phase (Gu et al., 1993) and CHO cells arrested at the G1 phase showed higher productivity (Fussenegger et al., 1997). Miller et al. (1990)
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found that gene transfers using retrovirus vectors occurred only in cells replicating actively at the time of infection, and that the efficiency of infection with retroviral virions was cell cycle dependent (Springett et al., 1989). In the case of the BES, TN-368 cells synchronized at the S phase were more susceptible to Autographa californica nucleopolyhedrovirus (AcNPV) infection than cells exposed at G2/M phase (Lynn and Hink, 1978). The sensitivity of infection in Sf9 cells was also affected by distribution of the cell cycle (Kioukia et al., 1995). The effect of AcNPV infection on host cell cycles has been known (Braunagel et al., 1998; Ikeda and Kobayashi, 1999; Prikhod’ko and Miller, 1998), but it is not obvious how host cell cycles participate in the virus replication or recombinant gene expression in the BES. Therefore, if the effects of cell cycle on recombinant gene expression and infection yield were clarified, the productivity of the recombinant protein and virus would increase. In this study, we investigate and clarify the effect of host cell cycle in the BES on recombinant gene expression and virus infection yield, and then discuss the changes that occur in the cell cycle, when the host cells are infected with AcNPV.
as a stock solution. The cell cycle of Sf9 cells was arrested at the M phase by incubation for 16 h in a TNM-FH medium containing 10 mg ml − 1 nocodazole (Braunagel et al., 1998). The release of cells from the nocodazole blockade was accomplished by washing them two times with a drug-free medium, followed by incubation in the fresh medium.
2.3. Infection Sf9 cells were infected at a multiplicity of infection (MOI) of 10. To examine the effect of cell cycle on GFPuv expression, cells were infected with 6GEPuv at different times in their cell cycles. Sf9 cells were released from the M phase and allowed to grow. They were then infected with 6GEPuv at a variety of times. This procedure is schematically shown in Fig. 1. If GFPuv expression was affected by the cell cycle, GFPuv expression would show different levels with different exposure times due to this procedure.
2. Materials and methods
2.1. Cell line and 6irus Sf9 cells (B825-01; Invitrogen, San Diego, CA, USA) derived from Spodoptera frugiperda were grown in 25 cm2 tissue culture flasks (Falcon) at 27 °C in TNM-FH medium (Sigma, St. Louis, MO, USA), supplemented with 0.35 g l − 1 NaHCO3, 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA) and 1% antibiotic– antimycotic (Gibco). Autographa californica nuclear polyhedrosis virus, inserted downstream of the polyhedral promoter with GFPuv gene (6GFPuv) (Cha et al., 1997) was used.
2.2. Nocodazole treatment Nocodazole (Sigma) dissolved in dimethyl sulfoxide at a concentration of 10 mg ml − 1 was used
Fig. 1. Schematic protocol of infection of Sf9 cells. All samples were allowed to enter the cell cycle simultaneously (t0). The virus was infected at different time (t0, t1, t2 shown as arrow pointing down), when each sample was positioned at different phases of cycle, as depicted by the heavy lines on the cycle next to each bar. The two arrows in each bar represent the beginning and end of the time period, t1 −t0 =t2 −t1 =3 h. The Sf9 cells were harvested at the time of arrow pointing up.
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2.4. Titer determination The virus samples were diluted serially as 10 − 3, 10 − 4, 10 − 5, 10 − 6 and 10 − 7. After mixing a 10 ml aliquot of each virus sample and 100 ml aliquots of the Sf9 cell suspension (1×105 cells ml − 1), each mixture of virus and cells was put into 12 wells of a 96-well plate. The plate was incubated at 27 °C for 7 days, and then, whether the cells were infected or not, was checked on the UV table. The titer of 6GFPuv was calculated using the standard method (Lucey et al., 1997).
2.5. Cell cycle analysis Sf9 cells in 25 cm2 tissue culture flasks were scraped by the cell scraper, collected into centrifuge tubes, washed two times in phosphatebuffered saline (PBS, pH 7.0) and fixed in an ice-cold 70% EtOH. After incubating at 4 °C for 30 min, the fixed cells were stored at − 20 °C until the commencement of cell cycle analysis. Prior to analysis, cells were washed two times in PBS and resuspended for 30 min in a propidium iodide (PI) (50 mg ml − 1 in PBS, 0.1% Triton X-100, 5 mg ml − 1 RNase A) solution at 37 °C (Braunagel et al., 1998). Cells stained with PI were filtered through a nylon membrane (350 meshes), and 2 ×104 cells were analyzed on a flow cytometer (EPICS ELITE, Beckman Coulter, Fullerton, CA, USA).
2.6. GFPu6 assay The GFPuv assay was performed by measuring fluorescence intensity using the flow cytometer. Cell populations infected with 6GFPuv were suspended in PBS and directly analyzed, where forward scatter (FSC), side scatter (SSC), and green fluorescence were recorded. From these data, cells were isolated from debris by their FSC vs SSC characteristics. Then the intracellular GFPuv fluorescence intensity of virus- and mock-infect 2 ×104 cells was measured (Farrell et al., 1998). Average intracellular GFPuv fluorescence intensity was defined as the difference in the intracellular GFPuv fluorescence intensity between virusand mock-infect cells as follows,
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Average intracellular GFPuv fluorescence intensity
%ni = 1(Ii × Xi,virus) (− )= %ni = 1Xi,virus
%ni = 1(Ii × Xi,mock) − , %ni = 1Xi,mock
where I, Xvirus and Xmock denote fluorescence intensity, virus-infected and mock-infected cells, respectively. The n indicates numbers of measured cell (n=20,000 cells).
2.7. Immunohistochemistry and confocal microscopy To measure the virus infection yield, the DNA binding protein (DBP) in infected cells was detected by immunochemical method. The DBP is expressed in the cells infected by AcNPV in an immediate early period, which is localized at the viral replication center and plays a role in the viral replication (Okano et al., 1999). Virus- or mock-infected Sf9 cells were fixed for 10 min with 2% formalin in PBS, washed three times with PBS, and permeabilized for 2 min in cold acetone (− 20 °C). The cells were rehydrated with PBS, blocked with 1% FBS in PBS for 1 h, and then subjected to antibody treatments. Localization of antigen was confirmed after incubation of the cells with rabbit anti-DBP serum (1:100 dilution with 1% FBS in PBS) for 1 h at room temperature. After the incubation with the appropriate primary antibody, cells were washed four times (5 min per wash) with PBS and the treated with the appropriate secondary antibody, rhodamine-conjugated goat anti-rabbit IgG (1:200 dilution with 1% FBS in PBS) for 1 h at room temperature. After five washes with PBS (5 min per wash), the cells were mounted with the mountant [10% v/v of PBS (0.01 M H3PO4 pH 7.4 in 0.15 M NaCl), 90% v/v glycerol containing 100 mg of p-phenylenediamine, pH 8.0] and covered with a coverslip. The edges of the coverslip were sealed with clear nail polish. The excitation and emission wavelengths for rhodamine were 550 and 580 nm, respectively. The expressed DBP was detected using a confocal scanning laser microscope (CSLM) (LSM 410, Carl Zeiss Co., Germany). Infection yield was defined as the percentage of DBP expressed cell number to total number of virus-infected cells.
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cells progressed from G1 to S phase, but cell cycle of virus-infected cells was arrested at S phase after 12 h post-infection and remained at the S phase. At 24 h post-infection, the cell cycles of mock-infected cells began to progress to the G2/M phase. However, in the case of virus-infected cells, the
Fig. 2. Cell cycle distribution of Sf9 cells. The cell cycle was synchronized at higher than 80% and was measured every 3 h post-release from block. The black, gray, white bars indicate the percentage of cells in G1, S, and G2/M phases, respectively.
3. Results
3.1. Cell cycle progression of infected Sf9 cells The Sf9 cells were synchronized in M phase by nocodazole treatment. After their release from M phase block, the cell cycle was measured every 3 h (Fig. 2). At 0 h post-release, more than 80% of the cells were synchronized in M phase. During the first 9 h post-release, the cell population in M phase decreased and that in G1 phase increased to 60% of cells. From 9 to 12 h post-release, cell cycles progressed from G1 to S phase, and more than 50% of the cells were in S phase at 15 h post-release. After 15 h post-release, the cell population in S phase decreased and that of G2/M phase began to increase. From these results, the doubling time of each cell was estimated to be 18 –21 h and the G1, S and G2/M phases were estimated to be 6, 6 and 6 h long, respectively under this condition. This is similar to previously published results (Braunagel et al., 1998). To examine the effect of 6GFPuv infection on progression of the cell cycle phase distribution, the Sf9 cells were partially synchronized in M, G1, and S phases and infected with 6GFPuv. At 0 h post-release, more than 70% of Sf9 cells were synchronized at M phase (Fig. 3A and B). During the first 8 h postinfection, the cell cycles progressed from M to G1 phase. From 8 to 18 h post-infection, the cell cycles of mock-infected
Fig. 3. Sf9 cells were synchronized at M phase with nocodazole treatment. After releasing from block, cells were harvested from the culture at the indicated time and measured cell cycle. A, C, and E denote DNA histograms of mock-infected cells, while B, D, and F denote DNA histograms of virus-infected cells. DNA histograms at 0 h post-release are A and B, those at 9 h post-release are C and D, and those at 15 h post-release are E and F, respectively.
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Fig. 4. Intracellular GFPuv fluorescence intensity of mock-infected (solid line) and virus-infected cells (dotted line).
histogram was broad and cells with higher than 4n DNA contents appeared. Similarly when Sf9 cells with more than 60% at G1 phase and more than 70% at S phase were infected, respectively, the progression of cell cycle was measured as shown in Fig. 3C– F. In the case of G1 phase-infection, there was no difference in cell cycle until post-infection time of 4 h, but the cell cycle of virus-infected cell began to be arrested at S phase from 8 h of post-infection, while that of mock-infected cells progressed. Finally the cell cycle of virus-infected cells was arrested at S phase with higher than 4n DNA content (Fig. 3C and D). In the case of S phase-infection, the cell cycle of virus-infected cell was arrested at G2/M phase after infection time of 4 h (Fig. 3F). It is clear that when the Sf9 cells are infected by baculovirus the cell cycle is finally arrested at the S or G2/M phase at 4– 12 h post-infection. However, cells with higher than 4n DNA content appeared at 18–24 h post-infection because of viral DNA replication.
3.2. Effect of cell cycle on GFPu6 expression To examine the effect of cell cycle on GFPuv expression, the Sf9 cells were infected at different cell cycle phases, and the intracellular GFPuv fluorescence intensity at early stage (before 24 h post-infection) was measured. This was because
either the cell cycles of infected cells were arrested at S or G2/M phase by 4–12 h post-infection (see Fig. 3) or secondary infection occurred after 18– 24 h post-infection. Difference in the intracellular GFPuv fluorescence intensity between mock- and virus-infected cells is shown in Fig. 4. The average fluorescence intensity of mock-infected cells was only 27.4 (solid line), while that of virus-infected cells was 124.8 (dotted line). This suggests that the average intracellular GFPuv fluorescence intensity of virus-infected cells is 97.4. Higher than 70% of the Sf9 cells were synchronized in M phase at 0 h post-release (Fig. 5A). During the first 6 h post-release, the cell population in G2/M phase decreased and that in G1 phase increased. At 6 h post-release, 77% of the Sf9 cells were in G1 phase. From 9 to 12 h post-release, cell cycles progressed from G1 to S phase, and 60% of Sf9 cells were in S phase. After 18 h post-release, cell cycles progressed to the G2/M phase in a less synchronous manner. The cells were infected at each point in the cell cycle, then average intracellular GFPuv expression at post-infection of 24 h as shown in Fig. 5B. The average intracellular GFPuv fluorescence intensity increased with the increase in the G1 phase fraction until 9 h post-release, but began to shrink after 12 h post-infection. The average intracellular GFPuv expression at the G1 phase-infection was 1.3-fold higher than that at the M phase. How-
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ever, after 24 h post-infection, it was difficult to investigate the effect of cell cycle on GFPuv expression because the cell cycles were no longer synchronized. From these results, it was obvious that since the GFPuv expression was corresponded to the fraction of G1 phase the G1 phaseinfection was the proper method for the GFPuv expression.
3.3. Effect of cell cycle on 6irus infection In order to investigate the virus infection at early stage the DBP expression was visualized by immunostaining infected Sf9 cells with anti-DBP antibodies and scanned with the CSLM. The temporal change in DBP expression is shown in Fig. 6A. The DBP began to be detected in the nucleus at 6 – 8 h post-infection (small circular foci) (A2 and A3). At 12 h post-infection, the expressed DBP was accumulated (A4) and transferred to near cell membrane (A5 and A6), and after 24 h
was decomposed inside cells. The infection yield increased with the increase in the MOI by 50, but remained at the highest infection yield, 70% in the higher MOI than 50 (Fig. 6B). The DBP foci per DBP expressed cells increased in the lower region than MOI= 10, but it remained at 9 foci per DBP expressed cell above higher MOI than 10 (Fig. 6C). These indicate that the infection yield is affected under the MOI of 50, but if the MOI is higher than 10 the DBP expression per infected cell is constant. Sf9 cells were partially synchronized in M, G1, and S phase, and infected with 6GFPuv. After 6 and 10 h post-infection the expressed DBP was detected to measure the infection yield as shown in Table 1. When cells were infected 6GFPuv the cells were average 82% of G2/M phase in run 1, average 57% of G1 phase in run 2, and average 70% of S phase in run 3. At 6 h post-infection, the infection yields in runs 1, 2 and 3 was 15, 27 and 23%, respectively. At 10 h post-infection, the infection yields in runs 1, 2 and 3 were 45, 66 and 67%, respectively. These results suggest that when the cell cycle is infected at G1 or S phase the infection yield is 1.5–1.8-fold higher than that at the G2/M phase-infection.
4. Discussion
Fig. 5. Effect of cell cycle on the intracellular GFPuv expression of Sf9 cell. (A) denotes distribution of cell cycle at infection. The black, gray, white bars indicate the percentage of cells in G1, S, and G2/M phases, respectively. The intracellular GFPuv fluorescence intensity was measured after harvesting the Sf9 cells at 24 h post-infection (B).
We investigated the Sf9 cell cycle progression after post-release from block (Fig. 3). When the Sf9 cells at G2/M, G1 or S phases were infected, the cells were arrested finally at S or G2/M phase and did not proceed further. These results suggest that the cell cycle of infected cells was finally arrested at S or G2/M phase by 4–12 h post-infection, regardless of when at any cell cycle Sf9 cells were infected. In addition, Sf9 cells with \ 4n DNA appeared after infection. Our results are coincident with other researches’ (Braunagel et al., 1998; Ikeda and Kobayashi, 1999). The Sf9 cells were infected at a variety of cell cycle distributions in order to investigate the influence of cell cycle on GFPuv expression. The average intracellular GFPuv fluorescence intensity was affected by the cell cycle and was corresponded to the profile of G1 phase (Fig. 5B). This result
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Fig. 6. DBP immunofluorescence images (A). When Sf9 cells were infected with 6GFPuv the DBP was detected at 6, 8, 12, 16, and 20 h as shown in A2 to A6, respectively. A1 denoted the image of mock-infected cells. Using various MOIs the infection yield (B) and the number of DBP foci per DBP expressed cell (C) were detected at 7 h post-infection.
suggests that the G1 phase of the cell cycle is more effective for the intracellular GFPuv expression. During the G1 phase, preparation of DNA synthesis and active RNA synthesis occur, and moreover, enzymes that are required for the synthesis of DNA and RNA may be rich. These may be the reasons that the infection at the G1 phase is suitable for the GFPuv expression. When the Sf9 cells were infected at G1 or S phase, the cell cycles were arrested at S or G2/M within 4 – 8 h post-infection, while in the case of G2/M phase-infection it took 8–12 h to be arrested at S phase. This suggests that in the cases of G1 or S infection, the viral gene expression begins from 4– 8 h post-infection, while in the case of G2/M phase-infection
from 8 to 12 h. This 4 h gap might be due to the low expression of GFPuv. The DBP detection in the BES was very useful to investigate the effect of cell cycle on the infection yield. The DBP is also present immediately after infection but decreases to low levels by 18 h post-infection, which indicates that the DBP functions at the early stage of infection as similar to IE2. The IE2 transcripts are present immediately after infection but decrease to low levels by 18 h post-infection, which means that the IE2 would be expected to function at the early stage of infection (Carson et al., 1991). When the cells were infected at G1 or S phase the infection yield was 1.5–1.8fold as high as that at G2/M phase (Table 1),
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which demonstrated that G1 or S phase-infections was effective for a recombinant protein production using the BES. Lynn and Hink (1978) showed that, when using a wild type virus, the infection efficiency at the S phase was better than at G2 phase. They speculated that cell membrane might be changed during the cell cycle progression. Kioukia et al. (1995) reported that during the S phase the increased DNA synthetic capacity would help the viral replication due to the availability of precursors and enzymes involved in viral replication. In this work it was clear that when the cells were infected at G1 or S phase, viral replication began 4 – 8 h faster than G2/M phase-infection. This may be one of the reasons higher infection yield at G1 or S phase-infection than G2/M phase-infection. In this work, we investigated the effects of cell cycle on the cell cycle progression, recombinant gene expression and infection yield in the BES. If a protein production is a main purpose using insect cells it may not be realistic to synchronize cell cycle. However, in the case of using the BES as a tool of protein production, it is very important to understand whether the cell cycle affects gene expression or not. The cell cycle of cells infected with AcNPV was arrested at the S or G2/M phase, at 4–12 h post-infection. The infection yield at the S or G1 phase was more sensitive than at G2/M phases, judging from both GFPuv expression and the DBP detection. In this work, when the insect cell is infected by baculovirus the changes in cell cycle progress and gene expression in the BES are shown, Table 1 Effect of cell cycle on infection yield in the BES Distribution of cell cycle on infection time (%)
Infection yield (%)
S
G2/M
PIT 6 h Run 1 5 Run 2 62 Run 3 19
11 22 73
84 16 8
15 27 23
PIT 10 h Run 1 6 Run 2 52 Run 3 23
14 21 67
80 27 10
45 66 67
G1
which may provide useful information to those who use the BES in effective recombinant protein production or virus production using the BES. Acknowledgements We are very grateful to Professor E. Bentley of the Department of Chem. Eng., University of Maryland for kindly providing recombinant AcNPV and 6GFPuv, and Dr N. Imai for providing anti-DBP serum. We would also like to thank to Dr K. Kamino, Marine Biotechnol. Inst. for kindly providing a flow cytometer. References Braunagel, S.C., Parr, R., Belyavskyi, M., Summers, M.D., 1998. Autographa californica nucleopolyhedro6irus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 244, 195 – 211. Carson, D.D., Summers, M.D., Guarino, L.A., 1991. Transient expression of Autographa californica nuclear polyhedrosis virus immediate-early gene, IE-N, is regulated by three viral elements. J. Virol. 65, 945 – 951. Cha, H.J., Pham, M.-Q., Rao, G., Bentley, W.E., 1997. Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. Biotechnol. Bioeng. 56, 239 – 247. Dee, K.U., Shuler, M.L., 1997. A mathematical model of the trafficking of acid-dependent enveloped viruses: application to the binding, uptake, and nuclear accumulation of baculovirus. Biotechnol. Bioeng. 54, 468 – 490. Farrell, P.J., Lu, M., Prevost, J., Brown, C., Behie, L., Iatrou, K., 1998. High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. Biotechnol. Bioeng. 60, 656 – 663. Fussenegger, M., Mazur, X., Bailey, J.E., 1997. A novel cytostatic process enhances the productivity of chinese humster ovary cells. Biotechnol. Bioeng. 55, 927 – 939. Gu, M.B., Todd, P., Kompala, D.S., 1993. Foreign gene expression (b-galactosidase) during the cell cycle phases in recombinant CHO cells. Biotechnol. Bioeng. 42, 1113 – 1123. Hink, F.W., Thomsen, D.R., Davidson, D.J.A., Meyer, L., Castellino, F.J., 1991. Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines. Biotechnol. Prog. 7, 9 – 14. Ikeda, M., Kobayashi, M., 1999. Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedro6irus. Virology 258, 176 – 188. Jesionowski, G.A., Ataai, M.M., 1997. An efficient medium for high protein production in the insect cell/baculovirus expression system. Biotechnol. Prog. 13, 355 – 360.
T. Saito et al. / Journal of Biotechnology 93 (2002) 121–129 Kioukia, N., Nienow, A.W., Emery, A.N., Al-Rubeai, M., 1995. Physiological and environmantal factors affecting the growth of insect cells and infection with baculovirus. J. Biotechnol. 38, 243 – 251. Licari, P., Bailey, J.E., 1991. Factors influencing recombinant protein yields in an insect cell-baculovirus expresson system: multiplicity of infection and intracellular protein degradation. Biotechnol. Bioeng. 37, 238 –246. Lucey, M.J., McColl, S.M., Manning, F.C.R., 1997. Simplification of titer determination for recombinant baculovirus by green fluorescent protein marker. BioTechniques 23, 782 – 786. Luckow, V.A., 1993. Baculovirus systems for the expression of human gene products. Curr. Opin. Biotechnol. 4, 564 –572. Luckow, V.A., Summers, M.D., 1988. Trends in the development of baculovirus expression vectors. Bio/Technology 6, 47– 55. Lynn, D.E., Hink, W.F., 1978. Infection of synchronized TN-368 cell cultures with alfalfa looper nuclear polyhedrosis virus. J. Invertebr. Pathol. 32, 1 –5. Miller, D.G., Adam, M.A., Miller, A.D., 1990. Gene transfer
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by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol. Cell. Biol. 10, 4239 – 4242. Okano, K., Mikhailov, V.S., Maeda, S., 1999. Colocalization of baculovirus IE-1 and two DNA-binding proteins, DBP and LEF-3, to viral replication factories. J. Virol. 73, 110 – 119. Prikhod’ko, E.A., Miller, L.K., 1998. Role of baculovirus IE2 and its ring finger in cell cycle arrest. J. Virol. 72, 684 – 692. Springett, G.L., Moen, R.C., Anderson, S., Blaese, R.M., 1989. Infection efficiency of T lymphocytes with amphotropic retroviral vectors is cell cycle dependent. J. Virol. 63, 3865 – 3869. Taticek, R.A., Shuler, M.L., 1997. Effect of elevated oxygen and glutamine levels on foreign protein production at high cell densities using the insect cell-baculovirus expression system. Biotechnol. Bioeng. 54, 142 – 152. Wang, P., Granados, R.R., Shuler, M.L., 1992. Studies on serum-free culture of insect cells for virus propagation and recombinant protein production. J. Invertebr. Pathol. 59, 46 – 53.
The effect of cell cycle on GFPuv gene expression in the baculovirus expression system Takehiko Saito a, Takashi Dojima b, Masaru Toriyama a, Enoch Y. Park a,* a
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka Uni6ersity, 836 Ohya, Shizuoka 422 -8529, Japan b Production Technology Laboratory, Kaken Pharmaceutical Co. Ltd., 301 Gensuke, Fujieda 426 -8646, Japan Received 26 January 2001; received in revised form 6 August 2001; accepted 20 August 2001
Abstract The effects of cell cycle on recombinant protein production and infection yield in the baculovirus-insect cell expression system (BES) were investigated. When, at any cell cycle phase, the host cell was infected by baculovirus, the cell cycle was finally arrested at the S or G2/M phase with 4n DNA. In the case of G1 or S phase-infection, cell cycle of virus-infected cells began to be arrested at S phase from 8 h post-infection or at G2/M phase from 4 h post-infection, respectively; while, in the case of M phase-infection, cell cycle was arrested at S phase after 12 h post-infection. When the host cell was infected at the G1 phase, average intracellular GFPuv fluorescence intensity was 1.3-fold higher than that at G2/M phase at 24 h post-infection. The GFPuv expression corresponded to the profile of the G1 cell cycle in the BES. Infection yield was measured by detection of intracellular DNA binding protein using immunohistochemical method within 7 h post-infection. The infection yield at G1 or S phase-infection was 1.5–1.8-fold higher than that at G2/M phase-infection. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Cell cycle; Sf9 cell; Baculovirus-insect cell expression system; GFPuv
1. Introduction The baculovirus expression system (BES) is widely used to express prokaryotic and eucaryotic proteins, because of its ease of use, high expression level (Luckow and Summers, 1988) and possibility of post-translational modifications, e.g. glucosylation and phosphorylation (Luckow, 1993). Many kinds of research into ways to improve the production of recombinant proteins * Corresponding author. Tel./fax: + 81-54-238-4887. E-mail address: [email protected] (E.Y. Park).
have been reported, including multiplicity of infection (MOI) (Licari and Bailey, 1991), cell line selection (Hink et al., 1991), culture condition (Jesionowski and Ataai, 1997; Taticek and Shuler, 1997; Wang et al., 1992) and an investigation of the infection process (Dee and Shuler, 1997). In mammalian cells, recombinant protein production and gene transfer using the virus were affected by host cell cycles. b-Galactosidase production rate in the S phase was higher than that in the G1 or G2/M phase (Gu et al., 1993) and CHO cells arrested at the G1 phase showed higher productivity (Fussenegger et al., 1997). Miller et al. (1990)
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found that gene transfers using retrovirus vectors occurred only in cells replicating actively at the time of infection, and that the efficiency of infection with retroviral virions was cell cycle dependent (Springett et al., 1989). In the case of the BES, TN-368 cells synchronized at the S phase were more susceptible to Autographa californica nucleopolyhedrovirus (AcNPV) infection than cells exposed at G2/M phase (Lynn and Hink, 1978). The sensitivity of infection in Sf9 cells was also affected by distribution of the cell cycle (Kioukia et al., 1995). The effect of AcNPV infection on host cell cycles has been known (Braunagel et al., 1998; Ikeda and Kobayashi, 1999; Prikhod’ko and Miller, 1998), but it is not obvious how host cell cycles participate in the virus replication or recombinant gene expression in the BES. Therefore, if the effects of cell cycle on recombinant gene expression and infection yield were clarified, the productivity of the recombinant protein and virus would increase. In this study, we investigate and clarify the effect of host cell cycle in the BES on recombinant gene expression and virus infection yield, and then discuss the changes that occur in the cell cycle, when the host cells are infected with AcNPV.
as a stock solution. The cell cycle of Sf9 cells was arrested at the M phase by incubation for 16 h in a TNM-FH medium containing 10 mg ml − 1 nocodazole (Braunagel et al., 1998). The release of cells from the nocodazole blockade was accomplished by washing them two times with a drug-free medium, followed by incubation in the fresh medium.
2.3. Infection Sf9 cells were infected at a multiplicity of infection (MOI) of 10. To examine the effect of cell cycle on GFPuv expression, cells were infected with 6GEPuv at different times in their cell cycles. Sf9 cells were released from the M phase and allowed to grow. They were then infected with 6GEPuv at a variety of times. This procedure is schematically shown in Fig. 1. If GFPuv expression was affected by the cell cycle, GFPuv expression would show different levels with different exposure times due to this procedure.
2. Materials and methods
2.1. Cell line and 6irus Sf9 cells (B825-01; Invitrogen, San Diego, CA, USA) derived from Spodoptera frugiperda were grown in 25 cm2 tissue culture flasks (Falcon) at 27 °C in TNM-FH medium (Sigma, St. Louis, MO, USA), supplemented with 0.35 g l − 1 NaHCO3, 10% fetal bovine serum (Gibco BRL, Rockville, MD, USA) and 1% antibiotic– antimycotic (Gibco). Autographa californica nuclear polyhedrosis virus, inserted downstream of the polyhedral promoter with GFPuv gene (6GFPuv) (Cha et al., 1997) was used.
2.2. Nocodazole treatment Nocodazole (Sigma) dissolved in dimethyl sulfoxide at a concentration of 10 mg ml − 1 was used
Fig. 1. Schematic protocol of infection of Sf9 cells. All samples were allowed to enter the cell cycle simultaneously (t0). The virus was infected at different time (t0, t1, t2 shown as arrow pointing down), when each sample was positioned at different phases of cycle, as depicted by the heavy lines on the cycle next to each bar. The two arrows in each bar represent the beginning and end of the time period, t1 −t0 =t2 −t1 =3 h. The Sf9 cells were harvested at the time of arrow pointing up.
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2.4. Titer determination The virus samples were diluted serially as 10 − 3, 10 − 4, 10 − 5, 10 − 6 and 10 − 7. After mixing a 10 ml aliquot of each virus sample and 100 ml aliquots of the Sf9 cell suspension (1×105 cells ml − 1), each mixture of virus and cells was put into 12 wells of a 96-well plate. The plate was incubated at 27 °C for 7 days, and then, whether the cells were infected or not, was checked on the UV table. The titer of 6GFPuv was calculated using the standard method (Lucey et al., 1997).
2.5. Cell cycle analysis Sf9 cells in 25 cm2 tissue culture flasks were scraped by the cell scraper, collected into centrifuge tubes, washed two times in phosphatebuffered saline (PBS, pH 7.0) and fixed in an ice-cold 70% EtOH. After incubating at 4 °C for 30 min, the fixed cells were stored at − 20 °C until the commencement of cell cycle analysis. Prior to analysis, cells were washed two times in PBS and resuspended for 30 min in a propidium iodide (PI) (50 mg ml − 1 in PBS, 0.1% Triton X-100, 5 mg ml − 1 RNase A) solution at 37 °C (Braunagel et al., 1998). Cells stained with PI were filtered through a nylon membrane (350 meshes), and 2 ×104 cells were analyzed on a flow cytometer (EPICS ELITE, Beckman Coulter, Fullerton, CA, USA).
2.6. GFPu6 assay The GFPuv assay was performed by measuring fluorescence intensity using the flow cytometer. Cell populations infected with 6GFPuv were suspended in PBS and directly analyzed, where forward scatter (FSC), side scatter (SSC), and green fluorescence were recorded. From these data, cells were isolated from debris by their FSC vs SSC characteristics. Then the intracellular GFPuv fluorescence intensity of virus- and mock-infect 2 ×104 cells was measured (Farrell et al., 1998). Average intracellular GFPuv fluorescence intensity was defined as the difference in the intracellular GFPuv fluorescence intensity between virusand mock-infect cells as follows,
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Average intracellular GFPuv fluorescence intensity
%ni = 1(Ii × Xi,virus) (− )= %ni = 1Xi,virus
%ni = 1(Ii × Xi,mock) − , %ni = 1Xi,mock
where I, Xvirus and Xmock denote fluorescence intensity, virus-infected and mock-infected cells, respectively. The n indicates numbers of measured cell (n=20,000 cells).
2.7. Immunohistochemistry and confocal microscopy To measure the virus infection yield, the DNA binding protein (DBP) in infected cells was detected by immunochemical method. The DBP is expressed in the cells infected by AcNPV in an immediate early period, which is localized at the viral replication center and plays a role in the viral replication (Okano et al., 1999). Virus- or mock-infected Sf9 cells were fixed for 10 min with 2% formalin in PBS, washed three times with PBS, and permeabilized for 2 min in cold acetone (− 20 °C). The cells were rehydrated with PBS, blocked with 1% FBS in PBS for 1 h, and then subjected to antibody treatments. Localization of antigen was confirmed after incubation of the cells with rabbit anti-DBP serum (1:100 dilution with 1% FBS in PBS) for 1 h at room temperature. After the incubation with the appropriate primary antibody, cells were washed four times (5 min per wash) with PBS and the treated with the appropriate secondary antibody, rhodamine-conjugated goat anti-rabbit IgG (1:200 dilution with 1% FBS in PBS) for 1 h at room temperature. After five washes with PBS (5 min per wash), the cells were mounted with the mountant [10% v/v of PBS (0.01 M H3PO4 pH 7.4 in 0.15 M NaCl), 90% v/v glycerol containing 100 mg of p-phenylenediamine, pH 8.0] and covered with a coverslip. The edges of the coverslip were sealed with clear nail polish. The excitation and emission wavelengths for rhodamine were 550 and 580 nm, respectively. The expressed DBP was detected using a confocal scanning laser microscope (CSLM) (LSM 410, Carl Zeiss Co., Germany). Infection yield was defined as the percentage of DBP expressed cell number to total number of virus-infected cells.
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cells progressed from G1 to S phase, but cell cycle of virus-infected cells was arrested at S phase after 12 h post-infection and remained at the S phase. At 24 h post-infection, the cell cycles of mock-infected cells began to progress to the G2/M phase. However, in the case of virus-infected cells, the
Fig. 2. Cell cycle distribution of Sf9 cells. The cell cycle was synchronized at higher than 80% and was measured every 3 h post-release from block. The black, gray, white bars indicate the percentage of cells in G1, S, and G2/M phases, respectively.
3. Results
3.1. Cell cycle progression of infected Sf9 cells The Sf9 cells were synchronized in M phase by nocodazole treatment. After their release from M phase block, the cell cycle was measured every 3 h (Fig. 2). At 0 h post-release, more than 80% of the cells were synchronized in M phase. During the first 9 h post-release, the cell population in M phase decreased and that in G1 phase increased to 60% of cells. From 9 to 12 h post-release, cell cycles progressed from G1 to S phase, and more than 50% of the cells were in S phase at 15 h post-release. After 15 h post-release, the cell population in S phase decreased and that of G2/M phase began to increase. From these results, the doubling time of each cell was estimated to be 18 –21 h and the G1, S and G2/M phases were estimated to be 6, 6 and 6 h long, respectively under this condition. This is similar to previously published results (Braunagel et al., 1998). To examine the effect of 6GFPuv infection on progression of the cell cycle phase distribution, the Sf9 cells were partially synchronized in M, G1, and S phases and infected with 6GFPuv. At 0 h post-release, more than 70% of Sf9 cells were synchronized at M phase (Fig. 3A and B). During the first 8 h postinfection, the cell cycles progressed from M to G1 phase. From 8 to 18 h post-infection, the cell cycles of mock-infected
Fig. 3. Sf9 cells were synchronized at M phase with nocodazole treatment. After releasing from block, cells were harvested from the culture at the indicated time and measured cell cycle. A, C, and E denote DNA histograms of mock-infected cells, while B, D, and F denote DNA histograms of virus-infected cells. DNA histograms at 0 h post-release are A and B, those at 9 h post-release are C and D, and those at 15 h post-release are E and F, respectively.
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Fig. 4. Intracellular GFPuv fluorescence intensity of mock-infected (solid line) and virus-infected cells (dotted line).
histogram was broad and cells with higher than 4n DNA contents appeared. Similarly when Sf9 cells with more than 60% at G1 phase and more than 70% at S phase were infected, respectively, the progression of cell cycle was measured as shown in Fig. 3C– F. In the case of G1 phase-infection, there was no difference in cell cycle until post-infection time of 4 h, but the cell cycle of virus-infected cell began to be arrested at S phase from 8 h of post-infection, while that of mock-infected cells progressed. Finally the cell cycle of virus-infected cells was arrested at S phase with higher than 4n DNA content (Fig. 3C and D). In the case of S phase-infection, the cell cycle of virus-infected cell was arrested at G2/M phase after infection time of 4 h (Fig. 3F). It is clear that when the Sf9 cells are infected by baculovirus the cell cycle is finally arrested at the S or G2/M phase at 4– 12 h post-infection. However, cells with higher than 4n DNA content appeared at 18–24 h post-infection because of viral DNA replication.
3.2. Effect of cell cycle on GFPu6 expression To examine the effect of cell cycle on GFPuv expression, the Sf9 cells were infected at different cell cycle phases, and the intracellular GFPuv fluorescence intensity at early stage (before 24 h post-infection) was measured. This was because
either the cell cycles of infected cells were arrested at S or G2/M phase by 4–12 h post-infection (see Fig. 3) or secondary infection occurred after 18– 24 h post-infection. Difference in the intracellular GFPuv fluorescence intensity between mock- and virus-infected cells is shown in Fig. 4. The average fluorescence intensity of mock-infected cells was only 27.4 (solid line), while that of virus-infected cells was 124.8 (dotted line). This suggests that the average intracellular GFPuv fluorescence intensity of virus-infected cells is 97.4. Higher than 70% of the Sf9 cells were synchronized in M phase at 0 h post-release (Fig. 5A). During the first 6 h post-release, the cell population in G2/M phase decreased and that in G1 phase increased. At 6 h post-release, 77% of the Sf9 cells were in G1 phase. From 9 to 12 h post-release, cell cycles progressed from G1 to S phase, and 60% of Sf9 cells were in S phase. After 18 h post-release, cell cycles progressed to the G2/M phase in a less synchronous manner. The cells were infected at each point in the cell cycle, then average intracellular GFPuv expression at post-infection of 24 h as shown in Fig. 5B. The average intracellular GFPuv fluorescence intensity increased with the increase in the G1 phase fraction until 9 h post-release, but began to shrink after 12 h post-infection. The average intracellular GFPuv expression at the G1 phase-infection was 1.3-fold higher than that at the M phase. How-
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ever, after 24 h post-infection, it was difficult to investigate the effect of cell cycle on GFPuv expression because the cell cycles were no longer synchronized. From these results, it was obvious that since the GFPuv expression was corresponded to the fraction of G1 phase the G1 phaseinfection was the proper method for the GFPuv expression.
3.3. Effect of cell cycle on 6irus infection In order to investigate the virus infection at early stage the DBP expression was visualized by immunostaining infected Sf9 cells with anti-DBP antibodies and scanned with the CSLM. The temporal change in DBP expression is shown in Fig. 6A. The DBP began to be detected in the nucleus at 6 – 8 h post-infection (small circular foci) (A2 and A3). At 12 h post-infection, the expressed DBP was accumulated (A4) and transferred to near cell membrane (A5 and A6), and after 24 h
was decomposed inside cells. The infection yield increased with the increase in the MOI by 50, but remained at the highest infection yield, 70% in the higher MOI than 50 (Fig. 6B). The DBP foci per DBP expressed cells increased in the lower region than MOI= 10, but it remained at 9 foci per DBP expressed cell above higher MOI than 10 (Fig. 6C). These indicate that the infection yield is affected under the MOI of 50, but if the MOI is higher than 10 the DBP expression per infected cell is constant. Sf9 cells were partially synchronized in M, G1, and S phase, and infected with 6GFPuv. After 6 and 10 h post-infection the expressed DBP was detected to measure the infection yield as shown in Table 1. When cells were infected 6GFPuv the cells were average 82% of G2/M phase in run 1, average 57% of G1 phase in run 2, and average 70% of S phase in run 3. At 6 h post-infection, the infection yields in runs 1, 2 and 3 was 15, 27 and 23%, respectively. At 10 h post-infection, the infection yields in runs 1, 2 and 3 were 45, 66 and 67%, respectively. These results suggest that when the cell cycle is infected at G1 or S phase the infection yield is 1.5–1.8-fold higher than that at the G2/M phase-infection.
4. Discussion
Fig. 5. Effect of cell cycle on the intracellular GFPuv expression of Sf9 cell. (A) denotes distribution of cell cycle at infection. The black, gray, white bars indicate the percentage of cells in G1, S, and G2/M phases, respectively. The intracellular GFPuv fluorescence intensity was measured after harvesting the Sf9 cells at 24 h post-infection (B).
We investigated the Sf9 cell cycle progression after post-release from block (Fig. 3). When the Sf9 cells at G2/M, G1 or S phases were infected, the cells were arrested finally at S or G2/M phase and did not proceed further. These results suggest that the cell cycle of infected cells was finally arrested at S or G2/M phase by 4–12 h post-infection, regardless of when at any cell cycle Sf9 cells were infected. In addition, Sf9 cells with \ 4n DNA appeared after infection. Our results are coincident with other researches’ (Braunagel et al., 1998; Ikeda and Kobayashi, 1999). The Sf9 cells were infected at a variety of cell cycle distributions in order to investigate the influence of cell cycle on GFPuv expression. The average intracellular GFPuv fluorescence intensity was affected by the cell cycle and was corresponded to the profile of G1 phase (Fig. 5B). This result
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Fig. 6. DBP immunofluorescence images (A). When Sf9 cells were infected with 6GFPuv the DBP was detected at 6, 8, 12, 16, and 20 h as shown in A2 to A6, respectively. A1 denoted the image of mock-infected cells. Using various MOIs the infection yield (B) and the number of DBP foci per DBP expressed cell (C) were detected at 7 h post-infection.
suggests that the G1 phase of the cell cycle is more effective for the intracellular GFPuv expression. During the G1 phase, preparation of DNA synthesis and active RNA synthesis occur, and moreover, enzymes that are required for the synthesis of DNA and RNA may be rich. These may be the reasons that the infection at the G1 phase is suitable for the GFPuv expression. When the Sf9 cells were infected at G1 or S phase, the cell cycles were arrested at S or G2/M within 4 – 8 h post-infection, while in the case of G2/M phase-infection it took 8–12 h to be arrested at S phase. This suggests that in the cases of G1 or S infection, the viral gene expression begins from 4– 8 h post-infection, while in the case of G2/M phase-infection
from 8 to 12 h. This 4 h gap might be due to the low expression of GFPuv. The DBP detection in the BES was very useful to investigate the effect of cell cycle on the infection yield. The DBP is also present immediately after infection but decreases to low levels by 18 h post-infection, which indicates that the DBP functions at the early stage of infection as similar to IE2. The IE2 transcripts are present immediately after infection but decrease to low levels by 18 h post-infection, which means that the IE2 would be expected to function at the early stage of infection (Carson et al., 1991). When the cells were infected at G1 or S phase the infection yield was 1.5–1.8fold as high as that at G2/M phase (Table 1),
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which demonstrated that G1 or S phase-infections was effective for a recombinant protein production using the BES. Lynn and Hink (1978) showed that, when using a wild type virus, the infection efficiency at the S phase was better than at G2 phase. They speculated that cell membrane might be changed during the cell cycle progression. Kioukia et al. (1995) reported that during the S phase the increased DNA synthetic capacity would help the viral replication due to the availability of precursors and enzymes involved in viral replication. In this work it was clear that when the cells were infected at G1 or S phase, viral replication began 4 – 8 h faster than G2/M phase-infection. This may be one of the reasons higher infection yield at G1 or S phase-infection than G2/M phase-infection. In this work, we investigated the effects of cell cycle on the cell cycle progression, recombinant gene expression and infection yield in the BES. If a protein production is a main purpose using insect cells it may not be realistic to synchronize cell cycle. However, in the case of using the BES as a tool of protein production, it is very important to understand whether the cell cycle affects gene expression or not. The cell cycle of cells infected with AcNPV was arrested at the S or G2/M phase, at 4–12 h post-infection. The infection yield at the S or G1 phase was more sensitive than at G2/M phases, judging from both GFPuv expression and the DBP detection. In this work, when the insect cell is infected by baculovirus the changes in cell cycle progress and gene expression in the BES are shown, Table 1 Effect of cell cycle on infection yield in the BES Distribution of cell cycle on infection time (%)
Infection yield (%)
S
G2/M
PIT 6 h Run 1 5 Run 2 62 Run 3 19
11 22 73
84 16 8
15 27 23
PIT 10 h Run 1 6 Run 2 52 Run 3 23
14 21 67
80 27 10
45 66 67
G1
which may provide useful information to those who use the BES in effective recombinant protein production or virus production using the BES. Acknowledgements We are very grateful to Professor E. Bentley of the Department of Chem. Eng., University of Maryland for kindly providing recombinant AcNPV and 6GFPuv, and Dr N. Imai for providing anti-DBP serum. We would also like to thank to Dr K. Kamino, Marine Biotechnol. Inst. for kindly providing a flow cytometer. References Braunagel, S.C., Parr, R., Belyavskyi, M., Summers, M.D., 1998. Autographa californica nucleopolyhedro6irus infection results in Sf9 cell cycle arrest at G2/M phase. Virology 244, 195 – 211. Carson, D.D., Summers, M.D., Guarino, L.A., 1991. Transient expression of Autographa californica nuclear polyhedrosis virus immediate-early gene, IE-N, is regulated by three viral elements. J. Virol. 65, 945 – 951. Cha, H.J., Pham, M.-Q., Rao, G., Bentley, W.E., 1997. Expression of green fluorescent protein in insect larvae and its application for heterologous protein production. Biotechnol. Bioeng. 56, 239 – 247. Dee, K.U., Shuler, M.L., 1997. A mathematical model of the trafficking of acid-dependent enveloped viruses: application to the binding, uptake, and nuclear accumulation of baculovirus. Biotechnol. Bioeng. 54, 468 – 490. Farrell, P.J., Lu, M., Prevost, J., Brown, C., Behie, L., Iatrou, K., 1998. High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector. Biotechnol. Bioeng. 60, 656 – 663. Fussenegger, M., Mazur, X., Bailey, J.E., 1997. A novel cytostatic process enhances the productivity of chinese humster ovary cells. Biotechnol. Bioeng. 55, 927 – 939. Gu, M.B., Todd, P., Kompala, D.S., 1993. Foreign gene expression (b-galactosidase) during the cell cycle phases in recombinant CHO cells. Biotechnol. Bioeng. 42, 1113 – 1123. Hink, F.W., Thomsen, D.R., Davidson, D.J.A., Meyer, L., Castellino, F.J., 1991. Expression of three recombinant proteins using baculovirus vectors in 23 insect cell lines. Biotechnol. Prog. 7, 9 – 14. Ikeda, M., Kobayashi, M., 1999. Cell-cycle perturbation in Sf9 cells infected with Autographa californica nucleopolyhedro6irus. Virology 258, 176 – 188. Jesionowski, G.A., Ataai, M.M., 1997. An efficient medium for high protein production in the insect cell/baculovirus expression system. Biotechnol. Prog. 13, 355 – 360.
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