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Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells
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Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells Joo-Hyoung Lee a,b , Sung-Min Lim b,c , Sun-Hye Park b,c , Jeong-Ki Min b,d , Gyun Min Lee a,∗∗ , Yeon-Gu Kim b,c,∗ a
Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea Biotherapeutics Translational Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea c Department of Bioprocess Engineering, UST, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea d Department of Biomolecular Science, UST, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea b
a r t i c l e
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Article history: Received 28 October 2016 Received in revised form 19 January 2017 Accepted 22 January 2017 Available online xxx Keywords: HEK293 cells EBNA-1/oriP dhfr/MTX Transient gene expression
a b s t r a c t In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (qp ), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fcfusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system. © 2017 Published by Elsevier Ltd.
1. Introduction Recombinant protein production systems for mammalian cell expression used in the biopharmaceutical industry have shown a remarkable improvement over the last few decades [1]. Stable gene expression has been predominantly used as a tool for the production of recombinant proteins. Because the development of stable cell lines is time consuming and labor intensive, transient gene expression (TGE) has proven to be an attractive alternative method for the rapid, simple, and cost-effective production of recombinant proteins in the early phases of development [2,3]. Mammalian cell lines such as Chinese hamster ovary (CHO), human embryonic kidney (HEK) 293, baby hamster kidney, and COS
∗ Corresponding author at: Biotherapeutics Translational Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. ∗∗ Corresponding author. E-mail addresses: [email protected] (G.M. Lee), [email protected] (Y.-G. Kim).
cell lines have been employed for TGE [4]. Among the mammalian cell lines, HEK293 and CHO are extensively used for large-scale TGE of therapeutic proteins [5]. Compared with the CHO cell-based TGE systems, the HEK293 cell-based TGE systems have shown better maintenance and higher transfection efficiencies of transgenes [6]. As a result, yields from HEK293 cells are usually higher than those from CHO cells in TGE [7]. Viral episomal vector systems have provided efficient strategies for increasing transgene expression [8]. In particular, the Epstein–Barr virus nuclear antigen-1 (EBNA-1), a human gammaherpesvirus that established latent infection in lymphocytes, has been investigated for the long-term expression of transgenes [9]. Thus, EBNA-1 in conjunction with the EB virus (EBV) latent origin of a replication (oriP) system is the most widely studied for episomal replication and retention in mammalian cells [8,10]. To support autonomous plasmid replication in HEK293 cells, HEK293 EBNA-1 (HEK293E) cells, which stably express EBNA-1, have been developed [11]. Previous studies have reported a significantly high expression level of transgenes owing to autonomous
http://dx.doi.org/10.1016/j.procbio.2017.01.020 1359-5113/© 2017 Published by Elsevier Ltd.
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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plasmid replication and retention of viral episomes in HEK293E cells compared with the level in HEK293 cells [12,13]. However, despite its superior transgene expression ability, the effect of EBNA1 expression level on the production of therapeutic proteins in HEK293 cells has not yet been elucidated. In this study, we investigated the effect of the EBNA-1 expression level on specific foreign protein productivity (qp ) in HEK293 cell-based TGE systems. To amplify the EBNA-1 expression, we used a dihydrofolate reductase (dhfr)-mediated gene amplification system. By employing this amplification system, we successfully developed EBNA-1-amplifed HEK293 cells with a higher expression of EBNA-1 than that in HEK293E cells.
2. Materials and methods 2.1. Construction of an EBNA-1-amplifying vector The EBNA-1 and internal ribosome entry site (IRES)-linked dhfr genes were prepared using a pCEP4 vector (Invitrogen) and pOptiVEC vector (Invitrogen), respectively. The PCR products of the EBNA-1 and IRES-linked dhfr genes were subcloned into a pcDNA3.1/zeo(+) vector (Invitrogen), yielding pcDNA-EBNA-1/dhfr. The expression of EBNA-1 and dfhr was driven by a CMV promoter, and a single cistron was enabled by IRES.
2.2. Cell line and culture maintenance The EBNA-1-amplified pools were established by the transfection of pcDNA-EBNA-1/dhfr into HEK293E cells, as previously described [14]. To screen for EBNA-1-amplified clones, a single clone selection was performed using a 96-well plate (Nunc). The single clone selection was performed from the EBNA-1amplified pool. Accordingly, representative selected single clones were EBNA-1-amplified single cells. Zeocin-resistant HEK293E cells (null) were constructed as control cells in a similar manner by transfection of the pcDNA3.1/zeo(+) vector. The representative clones were screened by Western blot analysis for EBNA-1 and adapted to grow in a suspension culture with a chemically defined medium (Expi293TM Expression Medium; Life Technologies). The cells were maintained with agitation at 110 rpm in a humidified 5% CO2 shaking incubator (INFORS HT Ecotron) at 37 ◦ C.
2.3. TGE pDR-OriP-Fc1, an Fc-fusion protein-expressing vector, was used for the TGE experiments [15]. Prior to transfection, cells in midexponential phase were centrifuged and resuspended to a density of 2.5 × 106 cells/mL into 125 mL Erlenmeyer flasks. Transfection with the ExpiFectamine 293TM reagent was performed according to the manufacturer’s instructions. The transfected cells were incubated and agitated at 110 rpm in a humidified 5% CO2 shaking incubator at 37 ◦ C. Samples were taken at the indicated days to determine the viable cell concentration and viability. After centrifugation, the culture supernatants were aliquoted and kept frozen at −70 ◦ C for subsequent analyses.
2.5. Western blot analysis Total cellular protein was evaluated by Western blot analysis, as previously described [16]. In brief, equal amounts of the cell lysates were loaded on 4%–12% Bis-Tris NuPAGE gel with non-reducing conditions. Antibodies used for the analysis were anti-EBV EBNA-1 (Santa Cruz Biotechnology) and anti-ˇ-actin antibody (Sigma). 2.6. Transfection efficiency Cells were transfected with the green fluorescent protein (GFP) green fluorescent protein (GFP)-expressing vector and harvested at 48 h post-transfection (hpt). Cells were then washed and resuspended in cold phosphate-buffered saline (PBS), and their fluorescent intensities were determined with a Guava EasyCyteTM HT system (Millipore), according to the manufacturer’s instructions. 2.7. Quantitative Real Time PCR (qRT-PCR) analysis The total DNA and RNA from each individual clone were isolated with the MiniBEST universal genomic DNA extraction kit and MiniBEST universal RNA extraction kit (TaKaRa), respectively, according to the manufacturer’s instructions. The isolated total DNA was digested by DpnI to cleavage plasmid DNA extracted from dam+ Escherichia coli [17,18]. A qRT-PCR analysis was performed as previously described [11]. All samples were normalized for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. The primer sequences used for qRT-PCR are listed in Table S1. 3. Results and discussion 3.1. Development of the EBNA-1-amplified HEK293 cells Clonal variation for amplified clones is an important issue when investigating the effect of gene expression in recombinant cells developed using a dhfr/methotrexate (MTX) gene amplification system. To avoid this issue, null-transfected cells (null pool) and EBNA-1-transfected cells (EBNA-1-amplified pool) were pooled and maintained in the selection medium with MTX. In general, cell lines developed by pool selection are unstable because they comprise individual clones with different growth characteristics. To confirm the EBNA-1 amplification, a Western blot analysis for EBNA-1 amplification in the null and EBNA-1-amplified pools was performed. Fig. 1A shows that the EBNA-1 expression level in the EBNA-1amplified pool was significantly higher than that in the null pool and its expression level was sustained during the pool selection. The application of the dhfr/MTX gene amplification system for developing EBNA-1-expressing HEK293 cells in this study could overcome the issue of a relatively low expression level of EBNA-1 in HEK293E cells. To evaluate the effect of EBNA-1 amplification on Fc-fusion protein production, null and EBNA-1-amplified pools were assessed by transient transfection with an Fc-fusion protein-producing vector. Compared with the control culture in the null pool, a 39.7% (P < 0.05) increase in Fc-fusion protein production was achieved in the EBNA-1-amplified pool (Fig. 1B). These results indicate that EBNA-1 amplification could improve Fc-fusion protein production in an HEK293 cell-based TGE system.
2.4. Cell concentration, viability, and Fc-fusion protein assay ®
Cell concentration and viability were estimated using a Vi-CELL XR analyzer (Beckman Coulter) with the trypan blue dye exclusion method. The secreted Fc-fusion protein concentration was quantified using a Cedex bio analyzer (Roche), according to the manufacturer’s instructions.
3.2. Relationship between EBNA-1 expression and qp To determine the relationship between the EBNA-1 expression level and qp , single clone selection in an EBNA-1-amplified pool was performed to screen the clones that stably amplified EBNA1 by Western blot analysis. Among the screened clones, 17 clones
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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Relative EBNA-1 expression Fig. 2. Relationship between the EBNA-1 expression level and qp in 17 selected clones. The expression level of EBNA-1 and qp were measured by Western blot analysis and Cedex Bio analyzer (Roche), respectively. The relative EBNA-1 expression was obtained by normalization with that in HEK293E cells. The EBNA-1 and qp (X, Y) pairs for each clone were plotted to calculate the correlation coefficient (R2 ). Error bars represent the standard deviations (n = 3).
with various EBNA-1 expression levels were selected from the measurement of band intensity by Quantity One intensity measurement program (Fig. S1). The 17 selected clones were then cultivated in 6-well plates in a serum-containing adherent culture mode and qp was estimated by transient transfection with a Fc-fusion proteinproducing vector. The qp was evaluated from a plot of the Fc-fusion protein concentration against the time integral values of the viable cell growth curve [19]. Fig. 2 shows the relationship of the relative EBNA-1 expression level and qp for the individual selected clones. The relative expression of EBNA-1 was obtained from the normalization of that for HEK293E cells. The qp was linearly correlated with the EBNA-1 expression, as indicated by the correlation coefficient (R2 = 0.7407). Accordingly, it was confirmed that the relative EBNA-1 expression level of HEK293 cells was positively correlated with qp . 3.3. TGE in EBNA-1-amplified HEK293 cells Before evaluating the usefulness of EBNA-1-amplified HEK293 cells in TGE systems, we investigated the effectiveness of cell
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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Fig. 4. The qp (A), relative mRNA expression (B), relative replicated DNA amount (C), and transfection efficiency (D) of EBNA-1-amplified clones during a TGE-based culture. (A) The qp was calculated using data collected from Day 1 to Day 3. (B and C) The relative mRNA expression and replicated DNA amount of the Fc-fusion protein were measured on Day 2 by normalizing that for HEK293 cells. (D) Transfection efficiency was analyzed at 48 hpt. Error bars represent the standard deviations (n = 3).
growth under a normal culture condition and not in TGE systems. Based on the cell growth in a serum-free suspension culture and qp in a transient transfection test (Fig. 2), we selected three clones (Eamp-68, E-amp-69, and E-amp-78). These clones were cultivated in a serum-free suspension culture without transient transfection. EBNA-1 amplification did not significantly affect the cell growth and viability profiles (Fig. S2). It was also found that metabolite utilization of glucose, lactate, glutamine, and ammonia were not affected by EBNA-1 amplification (data not shown). To determine the effect of EBNA-1 amplification on Fc-fusion protein production in TGE systems, the EBNA-1-amplified clones were transfected with an Fc-fusion protein-producing vector and cultivated in a serumfree suspension culture. Hyclone Cell BoostTM 5 (0.25%) was added to the cultures once on Day 3. A TGE-based culture of HEK293E cells was also obtained as control. Fig. 3 shows typical cell growth, viability, and Fc-fusion protein production during the TGE-based culture of EBNA-1-amplified clones. Although cell growth profiles varied among the cell lines, the pattern of viability decreased was not significantly affected by EBNA-1 amplification (Fig. 3A and B). The maximum Fc-fusion protein production in a TGE-based culture with three EBNA-1amplified clones was much higher than that in a culture with HEK293E cells (Fig. 3C). In particular, a 2.0-fold increase in the maximum Fc-fusion protein production (497 ± 51 mg/L) was achieved in E-amp-68 on Day 5 with a viability of more than 60%. These results
confirmed that EBNA-1-amplified HEK293 cells can be useful hosts for the TGE of therapeutic proteins. 3.4. Determination of molecular characteristics in EBNA-1-amplified HEK293 cells To determine the changes in the molecular characteristics by EBNA-1 amplification, the level of mRNA expression and amount of replicated DNA were measured in a TGE-based culture (Fig. 4). The episomal plasmid replication was detected by resistance to cleavage by DpnI, which requires recognition site methylated for activity. While the isolated plasmid DNA extracted from dam+ Escherichia coli was cleaved by DpnI, the replicated DNA in mammalian cells was resistant to DpnI cleavage [17,18]. The relative level of mRNA expression and the amount of replicated DNA for EBNA-1-amplified clones were compared with those from HEK293E cells on Day 2. Fig. 4A shows the qp calculated using data obtained from Day 1 to Day 3. Consistent with the results of the Fc-fusion protein production, only the TGE-based culture with the E-amp-68 and E-amp-78 significantly increased the qp by approximately 3.2- and 2.8-fold (P < 0.05) compared with the control culture. Fig. 4B shows the relative mRNA expression levels of EBNA-1-amplified clones on Day 2. Although the mRNA level was increased on Day 2, it was significantly decreased on Day 3 (data not shown). The qp was related to the mRNA expression level for Fc-fusion protein in all EBNA-1amplified clones. The mRNA expression level in E-amp-68 cells was
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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approximately 3.9-fold higher than that in HEK293E cells on Day 2. Fig. 4C shows the relative replicated DNA amount of EBNA-1amplified clones on Day 2. As previously described [17,18], we also observed that the episomally replicated DNA declined after reaching maximum levels on Day 2 (data not shown). A slightly higher amount of replicated DNA was achieved in E-amp-68. The different degree of enhancement in the level of mRNA expression and amount of replicated DNA may be associated with another effect of EBNA-1 on nuclear import of expression vector [2]. To evaluate the changes of transfection efficiency by EBNA-1 amplification, a GFP-expressing vector was transiently transfected into the HEK293E cells and EBNA-1-amplified clones. The transfection efficiency was determined as the percentage of GFP-positive cells in the total set of transfected cells and assessed at 48 hpt. Fig. 4D shows the results of the transfection efficiency in EBNA-1amplified clones. The EBNA-1-amplified clones and HEK293E cells did not show any significant differences in transfection efficiency, indicating that EBNA-1 amplification did not affect the transfection efficiency in HEK293 cells. Taken together, we concluded that an increased qp by EBNA-1 amplification mainly results from an enhancement in the amount of replicated DNA and level of mRNA expression but not from an improved transfection efficiency. Several approaches have been investigated such as optimization of transfection process, expression vector engineering, and culture strategies to achieve a high level of transgene expression in mammalian cell-based TGE systems [20–22]. In TGE system, plasmid copies are diluted during cell division, which results in a decrease in the recombinant protein productivity toward the end of the culture [17,18]. In a previous report, the regulation of transgene expression depends on the EBNA-1 expression level and oriP sequence [23]. In contrast to the noticeable effectiveness of EBNA-1 expression level on transgene expression in this study, Pham et al. (2003) reported that there was no significant correlation between EBNA-1 expression levels and transgene expression. This conflicting result may be because of a different range of EBNA-1 expression level with a different expression system, EBNA1-expressing vector, and EBNA-1-amplifying vector. In this study, we successfully used the dhfr/MTX gene amplification system to establish the EBNA-1-amplified HEK293 cells, thereby solving the issue of limited expression of EBNA-1 encountered in the use of an EBNA-1-expressing vector. Although we had to overcome the issue of a low expression level of EBNA-1 in HEK293E cells, EBNA-1 expression in EBNA-1amplified HEK293 cells was not linearly amplified by increasing the MTX concentration. It was inferred that the existence of dhfr in HEK293E cells hampers the dhfr/MTX gene amplification system, as previously described [24]. Thus, the development of dhfr-deficient HEK293 cells may be useful for an efficient dhfr/MTX gene amplification. In conclusion, EBNA-1 amplification in HEK293 cells enhances qp by enhancing the amount of replicated DNA and level of mRNA expression. Furthermore, the potential of EBNA-1-amplified HEK293 cells for therapeutic protein production by a TGE system was demonstrated in this study.
Acknowledgments This research was supported in part by a grant from KRIBB Initiative Program, a grant from the National Research Foundation of Korea funded by the Ministry of Science, Information & Communication Technology and Future Planning (grants NRF-2012M3A9C6050331), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (HO13C0002).
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Short communication
Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells Joo-Hyoung Lee a,b , Sung-Min Lim b,c , Sun-Hye Park b,c , Jeong-Ki Min b,d , Gyun Min Lee a,∗∗ , Yeon-Gu Kim b,c,∗ a
Department of Biological Sciences, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea Biotherapeutics Translational Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea c Department of Bioprocess Engineering, UST, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea d Department of Biomolecular Science, UST, 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 28 October 2016 Received in revised form 19 January 2017 Accepted 22 January 2017 Available online xxx Keywords: HEK293 cells EBNA-1/oriP dhfr/MTX Transient gene expression
a b s t r a c t In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (qp ), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fcfusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system. © 2017 Published by Elsevier Ltd.
1. Introduction Recombinant protein production systems for mammalian cell expression used in the biopharmaceutical industry have shown a remarkable improvement over the last few decades [1]. Stable gene expression has been predominantly used as a tool for the production of recombinant proteins. Because the development of stable cell lines is time consuming and labor intensive, transient gene expression (TGE) has proven to be an attractive alternative method for the rapid, simple, and cost-effective production of recombinant proteins in the early phases of development [2,3]. Mammalian cell lines such as Chinese hamster ovary (CHO), human embryonic kidney (HEK) 293, baby hamster kidney, and COS
∗ Corresponding author at: Biotherapeutics Translational Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. ∗∗ Corresponding author. E-mail addresses: [email protected] (G.M. Lee), [email protected] (Y.-G. Kim).
cell lines have been employed for TGE [4]. Among the mammalian cell lines, HEK293 and CHO are extensively used for large-scale TGE of therapeutic proteins [5]. Compared with the CHO cell-based TGE systems, the HEK293 cell-based TGE systems have shown better maintenance and higher transfection efficiencies of transgenes [6]. As a result, yields from HEK293 cells are usually higher than those from CHO cells in TGE [7]. Viral episomal vector systems have provided efficient strategies for increasing transgene expression [8]. In particular, the Epstein–Barr virus nuclear antigen-1 (EBNA-1), a human gammaherpesvirus that established latent infection in lymphocytes, has been investigated for the long-term expression of transgenes [9]. Thus, EBNA-1 in conjunction with the EB virus (EBV) latent origin of a replication (oriP) system is the most widely studied for episomal replication and retention in mammalian cells [8,10]. To support autonomous plasmid replication in HEK293 cells, HEK293 EBNA-1 (HEK293E) cells, which stably express EBNA-1, have been developed [11]. Previous studies have reported a significantly high expression level of transgenes owing to autonomous
http://dx.doi.org/10.1016/j.procbio.2017.01.020 1359-5113/© 2017 Published by Elsevier Ltd.
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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plasmid replication and retention of viral episomes in HEK293E cells compared with the level in HEK293 cells [12,13]. However, despite its superior transgene expression ability, the effect of EBNA1 expression level on the production of therapeutic proteins in HEK293 cells has not yet been elucidated. In this study, we investigated the effect of the EBNA-1 expression level on specific foreign protein productivity (qp ) in HEK293 cell-based TGE systems. To amplify the EBNA-1 expression, we used a dihydrofolate reductase (dhfr)-mediated gene amplification system. By employing this amplification system, we successfully developed EBNA-1-amplifed HEK293 cells with a higher expression of EBNA-1 than that in HEK293E cells.
2. Materials and methods 2.1. Construction of an EBNA-1-amplifying vector The EBNA-1 and internal ribosome entry site (IRES)-linked dhfr genes were prepared using a pCEP4 vector (Invitrogen) and pOptiVEC vector (Invitrogen), respectively. The PCR products of the EBNA-1 and IRES-linked dhfr genes were subcloned into a pcDNA3.1/zeo(+) vector (Invitrogen), yielding pcDNA-EBNA-1/dhfr. The expression of EBNA-1 and dfhr was driven by a CMV promoter, and a single cistron was enabled by IRES.
2.2. Cell line and culture maintenance The EBNA-1-amplified pools were established by the transfection of pcDNA-EBNA-1/dhfr into HEK293E cells, as previously described [14]. To screen for EBNA-1-amplified clones, a single clone selection was performed using a 96-well plate (Nunc). The single clone selection was performed from the EBNA-1amplified pool. Accordingly, representative selected single clones were EBNA-1-amplified single cells. Zeocin-resistant HEK293E cells (null) were constructed as control cells in a similar manner by transfection of the pcDNA3.1/zeo(+) vector. The representative clones were screened by Western blot analysis for EBNA-1 and adapted to grow in a suspension culture with a chemically defined medium (Expi293TM Expression Medium; Life Technologies). The cells were maintained with agitation at 110 rpm in a humidified 5% CO2 shaking incubator (INFORS HT Ecotron) at 37 ◦ C.
2.3. TGE pDR-OriP-Fc1, an Fc-fusion protein-expressing vector, was used for the TGE experiments [15]. Prior to transfection, cells in midexponential phase were centrifuged and resuspended to a density of 2.5 × 106 cells/mL into 125 mL Erlenmeyer flasks. Transfection with the ExpiFectamine 293TM reagent was performed according to the manufacturer’s instructions. The transfected cells were incubated and agitated at 110 rpm in a humidified 5% CO2 shaking incubator at 37 ◦ C. Samples were taken at the indicated days to determine the viable cell concentration and viability. After centrifugation, the culture supernatants were aliquoted and kept frozen at −70 ◦ C for subsequent analyses.
2.5. Western blot analysis Total cellular protein was evaluated by Western blot analysis, as previously described [16]. In brief, equal amounts of the cell lysates were loaded on 4%–12% Bis-Tris NuPAGE gel with non-reducing conditions. Antibodies used for the analysis were anti-EBV EBNA-1 (Santa Cruz Biotechnology) and anti-ˇ-actin antibody (Sigma). 2.6. Transfection efficiency Cells were transfected with the green fluorescent protein (GFP) green fluorescent protein (GFP)-expressing vector and harvested at 48 h post-transfection (hpt). Cells were then washed and resuspended in cold phosphate-buffered saline (PBS), and their fluorescent intensities were determined with a Guava EasyCyteTM HT system (Millipore), according to the manufacturer’s instructions. 2.7. Quantitative Real Time PCR (qRT-PCR) analysis The total DNA and RNA from each individual clone were isolated with the MiniBEST universal genomic DNA extraction kit and MiniBEST universal RNA extraction kit (TaKaRa), respectively, according to the manufacturer’s instructions. The isolated total DNA was digested by DpnI to cleavage plasmid DNA extracted from dam+ Escherichia coli [17,18]. A qRT-PCR analysis was performed as previously described [11]. All samples were normalized for the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level. The primer sequences used for qRT-PCR are listed in Table S1. 3. Results and discussion 3.1. Development of the EBNA-1-amplified HEK293 cells Clonal variation for amplified clones is an important issue when investigating the effect of gene expression in recombinant cells developed using a dhfr/methotrexate (MTX) gene amplification system. To avoid this issue, null-transfected cells (null pool) and EBNA-1-transfected cells (EBNA-1-amplified pool) were pooled and maintained in the selection medium with MTX. In general, cell lines developed by pool selection are unstable because they comprise individual clones with different growth characteristics. To confirm the EBNA-1 amplification, a Western blot analysis for EBNA-1 amplification in the null and EBNA-1-amplified pools was performed. Fig. 1A shows that the EBNA-1 expression level in the EBNA-1amplified pool was significantly higher than that in the null pool and its expression level was sustained during the pool selection. The application of the dhfr/MTX gene amplification system for developing EBNA-1-expressing HEK293 cells in this study could overcome the issue of a relatively low expression level of EBNA-1 in HEK293E cells. To evaluate the effect of EBNA-1 amplification on Fc-fusion protein production, null and EBNA-1-amplified pools were assessed by transient transfection with an Fc-fusion protein-producing vector. Compared with the control culture in the null pool, a 39.7% (P < 0.05) increase in Fc-fusion protein production was achieved in the EBNA-1-amplified pool (Fig. 1B). These results indicate that EBNA-1 amplification could improve Fc-fusion protein production in an HEK293 cell-based TGE system.
2.4. Cell concentration, viability, and Fc-fusion protein assay ®
Cell concentration and viability were estimated using a Vi-CELL XR analyzer (Beckman Coulter) with the trypan blue dye exclusion method. The secreted Fc-fusion protein concentration was quantified using a Cedex bio analyzer (Roche), according to the manufacturer’s instructions.
3.2. Relationship between EBNA-1 expression and qp To determine the relationship between the EBNA-1 expression level and qp , single clone selection in an EBNA-1-amplified pool was performed to screen the clones that stably amplified EBNA1 by Western blot analysis. Among the screened clones, 17 clones
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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Relative EBNA-1 expression Fig. 2. Relationship between the EBNA-1 expression level and qp in 17 selected clones. The expression level of EBNA-1 and qp were measured by Western blot analysis and Cedex Bio analyzer (Roche), respectively. The relative EBNA-1 expression was obtained by normalization with that in HEK293E cells. The EBNA-1 and qp (X, Y) pairs for each clone were plotted to calculate the correlation coefficient (R2 ). Error bars represent the standard deviations (n = 3).
with various EBNA-1 expression levels were selected from the measurement of band intensity by Quantity One intensity measurement program (Fig. S1). The 17 selected clones were then cultivated in 6-well plates in a serum-containing adherent culture mode and qp was estimated by transient transfection with a Fc-fusion proteinproducing vector. The qp was evaluated from a plot of the Fc-fusion protein concentration against the time integral values of the viable cell growth curve [19]. Fig. 2 shows the relationship of the relative EBNA-1 expression level and qp for the individual selected clones. The relative expression of EBNA-1 was obtained from the normalization of that for HEK293E cells. The qp was linearly correlated with the EBNA-1 expression, as indicated by the correlation coefficient (R2 = 0.7407). Accordingly, it was confirmed that the relative EBNA-1 expression level of HEK293 cells was positively correlated with qp . 3.3. TGE in EBNA-1-amplified HEK293 cells Before evaluating the usefulness of EBNA-1-amplified HEK293 cells in TGE systems, we investigated the effectiveness of cell
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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Fig. 4. The qp (A), relative mRNA expression (B), relative replicated DNA amount (C), and transfection efficiency (D) of EBNA-1-amplified clones during a TGE-based culture. (A) The qp was calculated using data collected from Day 1 to Day 3. (B and C) The relative mRNA expression and replicated DNA amount of the Fc-fusion protein were measured on Day 2 by normalizing that for HEK293 cells. (D) Transfection efficiency was analyzed at 48 hpt. Error bars represent the standard deviations (n = 3).
growth under a normal culture condition and not in TGE systems. Based on the cell growth in a serum-free suspension culture and qp in a transient transfection test (Fig. 2), we selected three clones (Eamp-68, E-amp-69, and E-amp-78). These clones were cultivated in a serum-free suspension culture without transient transfection. EBNA-1 amplification did not significantly affect the cell growth and viability profiles (Fig. S2). It was also found that metabolite utilization of glucose, lactate, glutamine, and ammonia were not affected by EBNA-1 amplification (data not shown). To determine the effect of EBNA-1 amplification on Fc-fusion protein production in TGE systems, the EBNA-1-amplified clones were transfected with an Fc-fusion protein-producing vector and cultivated in a serumfree suspension culture. Hyclone Cell BoostTM 5 (0.25%) was added to the cultures once on Day 3. A TGE-based culture of HEK293E cells was also obtained as control. Fig. 3 shows typical cell growth, viability, and Fc-fusion protein production during the TGE-based culture of EBNA-1-amplified clones. Although cell growth profiles varied among the cell lines, the pattern of viability decreased was not significantly affected by EBNA-1 amplification (Fig. 3A and B). The maximum Fc-fusion protein production in a TGE-based culture with three EBNA-1amplified clones was much higher than that in a culture with HEK293E cells (Fig. 3C). In particular, a 2.0-fold increase in the maximum Fc-fusion protein production (497 ± 51 mg/L) was achieved in E-amp-68 on Day 5 with a viability of more than 60%. These results
confirmed that EBNA-1-amplified HEK293 cells can be useful hosts for the TGE of therapeutic proteins. 3.4. Determination of molecular characteristics in EBNA-1-amplified HEK293 cells To determine the changes in the molecular characteristics by EBNA-1 amplification, the level of mRNA expression and amount of replicated DNA were measured in a TGE-based culture (Fig. 4). The episomal plasmid replication was detected by resistance to cleavage by DpnI, which requires recognition site methylated for activity. While the isolated plasmid DNA extracted from dam+ Escherichia coli was cleaved by DpnI, the replicated DNA in mammalian cells was resistant to DpnI cleavage [17,18]. The relative level of mRNA expression and the amount of replicated DNA for EBNA-1-amplified clones were compared with those from HEK293E cells on Day 2. Fig. 4A shows the qp calculated using data obtained from Day 1 to Day 3. Consistent with the results of the Fc-fusion protein production, only the TGE-based culture with the E-amp-68 and E-amp-78 significantly increased the qp by approximately 3.2- and 2.8-fold (P < 0.05) compared with the control culture. Fig. 4B shows the relative mRNA expression levels of EBNA-1-amplified clones on Day 2. Although the mRNA level was increased on Day 2, it was significantly decreased on Day 3 (data not shown). The qp was related to the mRNA expression level for Fc-fusion protein in all EBNA-1amplified clones. The mRNA expression level in E-amp-68 cells was
Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020
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approximately 3.9-fold higher than that in HEK293E cells on Day 2. Fig. 4C shows the relative replicated DNA amount of EBNA-1amplified clones on Day 2. As previously described [17,18], we also observed that the episomally replicated DNA declined after reaching maximum levels on Day 2 (data not shown). A slightly higher amount of replicated DNA was achieved in E-amp-68. The different degree of enhancement in the level of mRNA expression and amount of replicated DNA may be associated with another effect of EBNA-1 on nuclear import of expression vector [2]. To evaluate the changes of transfection efficiency by EBNA-1 amplification, a GFP-expressing vector was transiently transfected into the HEK293E cells and EBNA-1-amplified clones. The transfection efficiency was determined as the percentage of GFP-positive cells in the total set of transfected cells and assessed at 48 hpt. Fig. 4D shows the results of the transfection efficiency in EBNA-1amplified clones. The EBNA-1-amplified clones and HEK293E cells did not show any significant differences in transfection efficiency, indicating that EBNA-1 amplification did not affect the transfection efficiency in HEK293 cells. Taken together, we concluded that an increased qp by EBNA-1 amplification mainly results from an enhancement in the amount of replicated DNA and level of mRNA expression but not from an improved transfection efficiency. Several approaches have been investigated such as optimization of transfection process, expression vector engineering, and culture strategies to achieve a high level of transgene expression in mammalian cell-based TGE systems [20–22]. In TGE system, plasmid copies are diluted during cell division, which results in a decrease in the recombinant protein productivity toward the end of the culture [17,18]. In a previous report, the regulation of transgene expression depends on the EBNA-1 expression level and oriP sequence [23]. In contrast to the noticeable effectiveness of EBNA-1 expression level on transgene expression in this study, Pham et al. (2003) reported that there was no significant correlation between EBNA-1 expression levels and transgene expression. This conflicting result may be because of a different range of EBNA-1 expression level with a different expression system, EBNA1-expressing vector, and EBNA-1-amplifying vector. In this study, we successfully used the dhfr/MTX gene amplification system to establish the EBNA-1-amplified HEK293 cells, thereby solving the issue of limited expression of EBNA-1 encountered in the use of an EBNA-1-expressing vector. Although we had to overcome the issue of a low expression level of EBNA-1 in HEK293E cells, EBNA-1 expression in EBNA-1amplified HEK293 cells was not linearly amplified by increasing the MTX concentration. It was inferred that the existence of dhfr in HEK293E cells hampers the dhfr/MTX gene amplification system, as previously described [24]. Thus, the development of dhfr-deficient HEK293 cells may be useful for an efficient dhfr/MTX gene amplification. In conclusion, EBNA-1 amplification in HEK293 cells enhances qp by enhancing the amount of replicated DNA and level of mRNA expression. Furthermore, the potential of EBNA-1-amplified HEK293 cells for therapeutic protein production by a TGE system was demonstrated in this study.
Acknowledgments This research was supported in part by a grant from KRIBB Initiative Program, a grant from the National Research Foundation of Korea funded by the Ministry of Science, Information & Communication Technology and Future Planning (grants NRF-2012M3A9C6050331), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (HO13C0002).
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Please cite this article in press as: J.-H. Lee, et al., Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells, Process Biochem (2017), http://dx.doi.org/10.1016/j.procbio.2017.01.020