Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

High-level and high-throughput recombinant protein production by transient transfection of... © 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells Yves Durocher*, Sylvie Perret and Amine Kamen Animal Cell Technology and Downstream Processing Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada Received July 16, 2001; Revised and Accepted November 11, 2001 ABSTRACT transient transfection of mammalian cells grown in monolayers can generate significant amounts of r-proteins (1–3) but scala- A scalable transfection procedure using polyethylen- bility of this process is limited by culture surface availability. imine (PEI) is described for the human embryonic The well established calcium phosphate precipitation tech- kidney 293 cell line grown in suspension. Green nique or the recently described cationic polymer polyethylen- fluorescent protein (GFP) and human placental imine (PEI) (4) provides cost effective vehicles for the secreted alkaline phosphatase (SEAP) were used as introduction of plasmid DNA into mammalian cells. A major reporter genes to monitor transfection efficiency and breakthrough has recently emerged for the fast production of productivity. Up to 75% of GFP-positive cells were milligram amounts of recombinant proteins when these gene transfer vehicles were shown to be effective for the large-scale obtained using linear or branched 25 kDa PEI. The transfection of mammalian cells grown in suspension culture 293 cell line and two genetic variants, either (5–7). expressing the SV40 large T-antigen (293T) or the For an optimal large-scale transient transfection and r-protein Epstein–Barr virus (EBV) EBNA1 protein (293E), were expression in mammalian cells, four key aspects need to be tested for protein expression. The highest expression taken into account, namely (i) the cell line, (ii) the expression level was obtained with 293E cells using the EBV vector, (iii) the transfection vehicle and (iv) the culture oriP-containing plasmid pCEP4. We designed the medium. The human 293 cell line is widely used for r-protein pTT vector, an oriP-based vector having an improved production as it offers many advantages such as high transfection cytomegalovirus expression cassette. Using this yields with most gene transfer vehicles, is easily grown in vector, 10- and 3-fold increases in SEAP expression suspension culture, and can be adapted to serum-free media. was obtained in 293E cells compared with pcDNA3.1 Moreover, two genetic variants, the 293E and 293T cell lines, and pCEP4 vectors, respectively. The presence of expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal ampli- serum had a positive effect on gene transfer and fication of plasmids containing the viral EBV (293E) or SV40 expression. Transfection of suspension-growing (293T) origins of replication. Thus, they are expected to cells was more efficient with linear PEI and was not increase r-protein expression levels by permitting more affected by the presence of medium conditioned for plasmid copies to persist in the transfected cells throughout the 24 h. Using the pTT vector, >20 mg/l of purified His- production phase (8). The second important issue for high level tagged SEAP was recovered from a 3.5 l bioreactor. r-protein expression is to use vectors with promoters that are Intracellular proteins were also produced at levels as highly active in the host cell line, such as the human cyto- high as 50 mg/l, representing up to 20% of total cell megalovirus (CMV) promoter (9). This promoter is particularly proteins. powerful in 293 cells where it has been shown to be strongly transactivated by the constitutively expressed adenovirus E1a protein (10). Moreover, a highly efficient expression cassette INTRODUCTION using this promoter was recently described that provides Mammalian cells are an established expression system in the adenovirus-mediated transgene expression levels reaching up biotechnology industry for the production of recombinant to 20% of total cell proteins (TCP) (11,12). The third aspect is proteins (r-proteins). In contrast to lower eukaryotes or related to gene transfer reagent efficacy. Even though many prokaryotes, mammalian cells provide active r-proteins that highly effective gene transfer reagents are commercially possess relevant post-translational modifications. However, in available, only a few are cost effective when considering order to obtain sufficient amounts of protein for structure/activity operations at the multi-liters scale. For large-scale transient analyses or high-throughput screenings, one needs to go transfection applications, these reagents should also be simple through the long and tedious process of stable transfectoma to use, effective with suspension-growing cells and have isolation and characterization. As an alternative, the small-scale minimal cytotoxic effects. PEI satisfies most of these criteria *To whom correspondence should be addressed. Tel: +1 514 496 6192; Fax: +1 514 496 6785; Email: yves.durocher@nrc.ca e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 2 OF 9 as it has high gene transfer activity in many cell lines while pCEP5 vector. The pTT vector was generated following displaying low cytotoxicity (4), is cost effective and efficiently deletion of the hygromycin (BsmI and SalI excision followed transfects suspension growing 293 cells (6). This polymer is by fill-in and ligation) and EBNA1 (ClaI and NsiI excision available as both linear and branched isoforms with a wide followed by fill-in and ligation) expression cassettes. The ColE1 range of molecular weights and polydispersities, these physico- origin (FspI–SalI fragment, including the 3′ end of β-lactamase chemical parameters being critical for efficient gene transfer ORF) was replaced with a FspI–SalI fragment from pcDNA3.1 activity (13). The last key aspect for efficient protein expression containing the pMB1 origin (and the same 3′ end of β-lactamase by transient transfection deals with the culture medium. Some ORF). A Myc-(His) C-terminal fusion tag was added to SEAP gene transfer reagents work only in serum-free media whereas (HindIII–HpaI fragment from pSEAP-basic) following in-frame others are insensitive to the presence of serum. Also, as the ligation in pcDNA3.1/Myc-His digested with HindIII and presence of cellular by-products in conditioned medium is EcoRV. All plasmids were amplified in Escherichia coli (DH5α) associated with poor transfection yield, it is often necessary to grown in LB medium and purified using MAXI prep columns perform a complete medium exchange prior to transfection. (Qiagen, Mississauga, Ontario, Canada). For quantification, However, this step does not satisfy the need for a robust large- plasmids were diluted in 50 mM Tris–HCl pH 7.4 and the scale transient transfection process. absorbances at 260 and 280 nm measured. Only plasmid preparations with A /A ratios between 1.75 and 2.00 were In this study, the model proteins, green fluorescent protein 260 280 used. (GFP) and secreted alkaline phosphatase (SEAP), were used to design an expression vector and establish transfection para- Small-scale transient transfections meters in order to reach high expression levels in suspension growing 293E cells using both linear and branched 25 kDa Three hours before transfection, cells were centrifuged and PEI. We also show that this technology is fully adapted for the resuspended in fresh HSFM supplemented with 1% BCS at a 6 –1 high-throughput production of r-proteins and will assuredly be density of 1.0 × 10 cells ml . Five hundred microliters, or useful for structure–function studies and high-throughput 10 ml, of cell suspension was distributed per well in a 12-well screening assays. plate, or in a 125 ml shaker flask, respectively. DNA was diluted in fresh serum-free HSFM (in a volume equivalent to one-tenth of the culture to be transfected), PEI was added, and MATERIALS AND METHODS the mixture immediately vortexed and incubated for 10 min at room temperature prior to its addition to the cells. Following a Chemicals 3 h incubation with DNA–PEI complexes, culture medium was A 25 kDa branched PEI was obtained from Aldrich completed to 1 ml (12-well plate) or 20 ml (shaker flask) by the (Milwaukee, WI) and 25 kDa linear PEI from Polysciences addition of HSFM supplemented with 1% BCS. –1 (Warrington, PA). Stock solutions (1 mg ml ) were prepared Transfection in bioreactors in water, neutralized with HCl, sterilized by filtration (0.22 µ m), aliquoted and stored at –80°C. A 3.5-l bioreactor containing 2.85 l of HSFM supplemented with 1% BCS was seeded with 293E cells to obtain a final cell Cell culture 5 –1 density of 2.5 × 10 ml . Twenty-four hours later, cells were Human embryonic kidney 293S (293) cells (14) and genetic transfected with 150 ml of a mixture of pTT/SEAP:pEGFP variants stably expressing EBNA1 (293E) (Invitrogen, plasmids (19:1, 3 mg total) and PEI (6 mg). Agitation was at Carlsbad, CA) or the large-T antigen (293T) (15) were adapted 70 r.p.m. using a helical ribbon impeller (16). Dissolved to suspension culture in low-calcium-hybridoma serum-free oxygen was maintained at 40% by surface aeration using a medium (HSFM) (14) supplemented with 1% bovine calf serum nitrogen/oxygen mixture (300 ml/min) and pH was maintained –1 (BCS), 50 µ g ml Geneticin (for 293E and 293T cells), 0.1% at 7.2 by addition of CO in the head space and sodium Pluronic F-68 (Sigma, Oakville, Ontario, Canada) and 10 mM bicarbonate [10% (w/v) in water] injection in the culture HEPES. For culture in bioreactors, HEPES was omitted from the medium. The same conditions were used for transfection in 14-l medium. Cells were cultured in Erlenmeyer flasks (50 or 125 ml) bioreactors. using 15–25% of the nominal volume at 110–130 r.p.m. Flow cytometry (Thermolyne’s BigBill orbital shaker; TekniScience Inc., Terre- bonne, Québec, Canada) under standard humidified conditions GFP was analyzed by flow cytometry using an EPICS Profile (37°C and 5% CO ). II (Coulter, Hialeah, FL) equipped with a 15-mW argon-ion laser. Only viable cells were analyzed for the expression of Vectors GFP. Data are representative of at least two independent The pIRESpuro/EGFP (pEGFP) and pSEAP basic vectors experiments. Error bars represent ±SEM of one experiment were obtained from Clontech (Palo Alto, CA), and pcDNA3.1, done in duplicate. pcDNA3.1/Myc-(His) and pCEP4 vectors were from Invitrogen. SEAP analysis The SuperGlo GFP variant (sgGFP) was from Q·Biogene (Carlsbad, CA). Construction of pCEP5 vector was as follows: Determination of SEAP activity was performed essentially as the CMV promoter and polyadenylation signal of pCEP4 were previously described by Durocher et al. (17). Briefly, culture removed by sequential digestion and self-ligation using SalI medium was diluted in water as required (typically 1/50 to 1/1000) and XbaI enzymes, resulting in plasmid pCEP4∆ . A BglII fragment and 50 µ l was transferred to a 96-well plate. Fifty microliters from pAdCMV5 (11) encoding the CMV5-poly(A) expression of SEAP assay solution containing 20 mM paranitrophenyl- cassette was ligated in BglII-linearized pCEP4∆ , resulting in phosphate (pNPP), 1 mM MgCl , 10 mM l-homoarginine and 2 PAGE 3 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 1 M diethanolamine pH 9.8 were then added and absorbance read at 410 nm at 1–2 min intervals at room temperature to determine pNPP hydrolysis rates. Data are representative of at least two independent experiments. Error bars represent ±SEM of one experiment done in duplicate. For the bioreactor run, error bars represent ±SEM of two SEAP measurements. Electrophoresis, western analyses and quantification Immunodetection of C-terminal Myc-(His) -tagged SEAP was done using the anti-Myc 9E10 antibody (Santa Cruz). For analysis of intracellular proteins, cells were solubilized in NuPAGE sample buffer (Novex) or extracted with lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Thesit and 0.5% sodium deoxycholate). Insoluble material was removed from lysates by centrifugation at 12 000 g at 4°C for 5 min. Concentrated NuPAGE buffer (4×) was added to cleared lysates. All samples were heated for 3 min at 95°C. Proteins were resolved on 4–12% Bis–Tris or 3–8% Tris–acetate NuPAGE gradient gels as recommended by the manufacturer. GFP and other non-tagged proteins were quantified relative to purified bovine serum albumin (BSA) following electrophoresis and Coomassie blue R250 staining using the Kodak Digital Science Image Station 440cf equipped with the Kodak Digital Science 1D image analysis software version 3.0 (Eastman Kodak, NY). RR1 was quantified by slot-blot relative to a homogeneity-purified RR1 standard detected by using a monoclonal anti-RR1 antibody. Other Myc-(His) -tagged proteins were quantified relative to purified SEAP-Myc-(His) . RESULTS Figure 1. Effect of DNA to PEI ratio on transfection efficiency. 293E cells were Transfection with linear and branched 25 kDa PEI transfected with linear (A) or branched (B) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations Preliminary results showed that linear and branched 25 kDa –1 (µ g ml ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 PEI were the most effective among various polymers tested (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt. (including branched 70 kDa, branched 50–100 kDa and branched 10 kDa; data not shown). Therefore, we optimized transfection of 293E cells with both linear or branched 25 kDa plasmid DNA up to 90 copies in cells expressing the EBNA1 PEI polymers using a plasmid encoding the enhanced GFP protein (19). We also generated the pCEP5 vector (Fig. 2A, (pEGFP). Transfections were performed using cells grown as left) by using an improved CMV expression cassette as monolayers in 12-well plates and GFP expression was measured described in the adenoviral transfer vector pAdCMV5 (20). 72 h later by flow cytometry. The effect of DNA to PEI ratios This expression cassette has been shown to confer very high on transfection efficiency is shown in Figure 1 using linear (A) levels of r-protein expression in 293 cells (12). The pCEP5 or branched (B) PEI. The indicated amounts of DNA and vector was further modified (Materials and Methods) to yield polymer are for one well containing 5 × 10 cells. Only 0.25 µ g the pTT vector (Fig. 2A, right) that is 4.6 kb smaller, hence of DNA was sufficient to reach a 50% transfection efficiency using linear PEI, whereas a minimum of 1.0 µ g was necessary providing more space for large cDNA cloning. The cDNA using the branched isoform. Transfection efficiencies of ∼70% encoding for the reporter protein SEAP was then cloned in were reached with both linear and branched polymers at each of these four vectors and its expression level monitored DNA:PEI (µ g:µ g) ratios of 1.0:1.5 and 1.5:2.0, respectively. following transient transfection in 293, 293T or 293E cells. As Increasing the amounts of both DNA and PEI did not lead to shown in Figure 2B, transfection of the 293T cell line with the higher transfection yield. SV40 ori-containing plasmid pcDNA3.1 did not translate into an increased transgene expression when compared with trans- Cell line and expression vectors fection of the parental 293 cells. However, transfection of 293E cells with the pCEP4 vector resulted in a 2–3-fold Two commercially available expression vectors containing increase in SEAP expression compared with transfection of 293 viral sequences allowing for episomal DNA replication in or 293T cells with the same vector. In addition, the use of the permissive cell lines were tested. The first vector, pcDNA3.1, contains the SV40 origin of replication that allows cellular pCEP5 vector further increased SEAP expression by a factor polymerases to replicate the DNA up to 10 000 copies in cells of 2–6-fold, depending on the cell line. Finally, the use of the expressing the large T antigen (18). The second vector, pCEP4, pTT vector in 293E cells resulted in a 33% increase in trans- contains the EBV origin of replication oriP that replicates gene expression compared with the pCEP5 vector. The overall e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 4 OF 9 Figure 2. Effect of cell lines and vectors on SEAP expression. (A) Genetic maps of pCEP5 (left) and pTT (right) vectors drawn to scale. The pCEP5 vector backbone is identical to pCEP4 vector except for the transgene expression cassette. Construction of the pTT vector is as described in Materials and Methods. TPL, tripartite leader; enh MLP, adenovirus major late promoter enhancer; SD, splice donor; SA, splice acceptor; DS, dyad symmetry; FR, family of repeats. (B) Cells were transfected with 1 µ g of DNA and 2 µ g of linear PEI and SEAP activity measured 72 hpt. The pEGFP plasmid (0.1 µ g) was also added in each condition to monitor for transfection efficiency and SEAP activities were normalized accordingly. Open boxes, pcDNA3.1/SEAP; hatched boxes, pCEP4/SEAP; gray boxes, pCEP5/SEAP; closed boxes, pTT/SEAP. SEAP expression level in 293E cells was 10-fold higher with BCS-supplemented HSFM. Shaker flask cultures were co-trans- the pTT vector compared with the pcDNA3.1 vector. fected with a mixture of pTT/SEAP:pEGFP (9:1) plasmids (pEGFP was added to monitor transfection efficiency). With Effect of serum both linear and branched PEI, SEAP accumulated in the culture medium for up to 96 hours post-transfection (hpt) (Fig. 4), but The effect of serum on transfection efficiency (GFP) and r-protein gene transfer and expression level were 50% higher using the production (SEAP) mediated by both linear and branched PEI was evaluated. Figure 3 shows that when transfection mixture was linear isoform. These results clearly demonstrate that linear, and to a lesser extent branched, PEI are effective for gene transfer added to cells in fresh 1% serum-containing medium, a 4–5-fold in suspension-growing cells. In addition, SEAP expression increase in SEAP activity was obtained compared with its levels obtained with suspension-growing cells using linear PEI addition to cells in serum-free medium. Increasing serum concentration up to 5% further improved PEI-mediated trans- were comparable with those obtained with adherent-growing fection efficiency and production. When transfection mixture cells. For all experiments reported below, only linear PEI was was added to cells in serum-free media followed 3 h later by used. serum addition to a concentration of 1% (0→1%), a 2-fold Our goal was to define a robust, simple and scalable trans- increase in transgene expression was obtained; however, this fection process. In order to reach these objectives, two steps level was only 50% of that obtained in 1% serum. had to be simplified: the 3 h incubation of DNA–PEI complexes with cells in a reduced culture volume, and the Process optimization for transfection in suspension medium change 3 h prior to transfection. The first step was We next evaluated gene transfer efficiency of both linear and performed with the assumption that it would promote inter- branched PEI on suspension-growing 293E cells in 1% action of the DNA–PEI complexes with the cells and thus PAGE 5 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 Figure 4. Transfection of suspension growing cells. Cells were resuspended in 6 –1 10 ml of fresh HSFM containing 1% BCS to a density of 1 × 10 ml in a 125 ml Erlenmeyer flask. Three hours later, 1 ml of the DNA–PEI complexes were added and the culture incubated for an additional 3 h. The volume was then completed to 20 ml with fresh culture medium. The DNA–PEI complexes were as follows: 40 µ g of linear or branched PEI was added to 1 ml of HEPES- supplemented HSFM containing 18 µ g of pTT/SEAP and 2 µ g of pEGFP or 27 µ g of pTT/SEAP and 3 µ g of pEGFP, respectively. Open symbols, linear PEI; closed symbols: branched PEI. observed when the transfection was carried out in medium conditioned for 24 h, indicating that medium exchange is not necessary. Transfection in bioreactors To demonstrate scalability of the process, a 3.5-l bioreactor Figure 3. Effect of serum on transgene expression. 293E cells were transfected culture was transfected with a mixture of pTT/SEAP:pEGFP with pTT/sgGFP (A) or pTT/SEAP (B) vectors using 1.0 µ g of DNA and plasmids (19:1). One hour later, a sample (25 ml) was withdrawn 2.0 µ g of linear PEI (hatched boxes) or 1.5 and 2.0 µ g of branched PEI (gray and transferred into a shaker flask as a control. In the bioreactor boxes) in fresh serum-free or serum-supplemented media. In one experiment (0→1%), cells were transfected in serum-free media and serum was added 3 h (Fig. 6A, unbroken lines), SEAP (circles) accumulated up to later to a final concentration of 1%. GFP-positive cells and SEAP activity were 144 hpt and then reached a plateau whereas accumulation measured 72 hpt. continued up to 216 hpt in the control shaker flask (dashed lines). The percentage of GFP-positive cells (squares) at 96 hpt reached 54 and 50% for the bioreactor and the shaker flask, increase transfection efficiency. The second was done according to reports showing a deleterious effect of conditioned medium on respectively. At the end of the culture, cell density was 4.1 and 6 –1 transfection efficiency (6,21). Whereas medium exchange is 4.7 × 10 ml with a viability of 62 and 72% for the bioreactor simple to perform on a small scale, this step represents a and shaker flask, respectively (Fig. 6B). Although viable cell significant hurdle at scales greater than a few liters. density was 25% lower in the bioreactor compared with the shaker flask, volumetric SEAP productivity was almost 2-fold The effect of cell density at time of transfection was first evaluated (Fig. 5A) by transfecting high density (hatched bars; higher. Similar results were systematically observed in five 6 –1 10 ml at 1 × 10 cells ml ) or low density cultures (gray bars; independent experiments (results not shown), indicating that 5 –1 the productivity of secreted proteins might be increased when 20 ml at 5 × 10 cells ml ) in shaker flasks. Three hours later, 5 –1 using a controlled environment. the high cell density flask was diluted to 5 × 10 cells ml with fresh medium, and GFP expression in both flasks monitored Purification of SEAP and production of other r-proteins 72 h later. This experiment showed that cell concentration prior to transfection could be omitted as only a slight decrease Purification of Myc-(His) -tagged SEAP harvested from the (<10%) in transfection efficiency and a 15% decrease in GFP bioreactor run (Fig. 6) by immobilized metal affinity chroma- expression level were observed when cells were transfected in tography (IMAC) is shown in Figure 7A. The left panel shows a larger culture volume. Coomassie blue-stained protein pattern from the culture We next evaluated the effect of conditioned medium on medium before loading on the column (lane 1), flow-through SEAP expression using suspension growing cells. For this (lane 2) and eluted material using 150 mM imidazole (lane 3). study, cells were seeded in shaker flasks at a density of 2.5 × The right panel shows immunodetection of SEAP in the same 5 –1 10 ml . Twenty-four hours later, transfection was performed fractions using anti-Myc antibody. This figure shows that all of with or without a complete medium exchange. As shown in the His-tagged SEAP was retained on the column whereas very Figure 5B, no significant difference in SEAP expression was few, if any, serum proteins bound to it (SEAP migrates with an e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 6 OF 9 Figure 6. Transient transfection in a 3.5-l bioreactor. (A) 293E cells were 5 –1 seeded at a density of 2.5 × 10 ml in 2.85 l of fresh HSFM supplemented with 1% BCS. Twenty-four hours later, the transfection mixture (6 mg of linear PEI added to 150 ml HSFM containing 2.85 mg pTT/SEAP and 150 µ g pEGFP plasmids) was added to the bioreactor (unbroken lines). One hour later, Figure 5. Effect of cell density and of conditioned medium. (A) Transfection 25 ml of culture was withdrawn from the bioreactor and transferred in a shake efficiency and relative total GFP expression (in percent) obtained following flask as a control (dashed lines). SEAP activity (circles) and GFP-positive cells 6 –1 transfection using standard conditions (hatched bars: 10 ml of cells at 1 × 10 ml (squares) were determined as described in Materials and Methods. (B) Growth followed by addition of 10 ml of fresh medium 3 h after transfection) or using curves (diamonds), viability (triangles) and yO (gray line) in the 3.5-l bioreactor 5 –1 cells at 5 × 10 ml in 20 ml of culture medium (gray bars). GFP was monitored (unbroken lines) and shaker flask (dashed lines). 72 hpt. Relative total GFP was obtained following multiplication of percent GFP-positive cells by the mean fluorescence intensity. (B) Cells were seeded 5 –1 in 20 ml of 1% BCS-supplemented HSFM at a density of 2.5 × 10 ml 24 h before transfection. The medium was then left unchanged (conditioned: open r-proteins were also obtained as shown in Figure 7C. In this circles) or replaced with 20 ml of fresh medium (closed circles). Three hours experiment, 293E cells were transfected with pTT plasmids later, cells were transfected by the addition of 2 ml of DNA–PEI complexes encoding for sgGFP (lane 1), herpes simplex virus ribonucleo- (20 µ g of pTT/SEAP and 40 µ g of linear PEI). tide reductase 1 (RR1, lane 2), mouse G (lane 5), human αq Kip1 p27 (lane 6), yeast pyruvate carboxylase (PYC, lane 7), 19K adenovirus E1B (lane 8), human hexokinase 1 (HK, lane 9) apparent molecular weight slightly higher than BSA). SEAP and human glucokinase (GK, lane 10). Three days after trans- quantification in the eluted fraction using the Lowry protein fection, cells were rinsed with PBS, solubilized in sample assay showed that ∼60 mg of His-tagged SEAP could be buffer (GFP, RR1 and G ) or extracted with lysis buffer αq recovered by IMAC from the 3-l bioreactor culture. As shown Kip1 19K (p27 , PYC, E1B , HK and GK), and proteins analyzed by in Figure 7B, high expression levels in bioreactor were also SDS–PAGE. Quantification of r-proteins shown in Figure 7 is obtained with other secreted r-proteins. Fourteen- (lanes 1, 3 summarized in Table 1. In the case of RR1, volumetric production and 4) or 3.5-liter (lane 2) bioreactors were transfected with was 50 mg/l, representing 20% of TCP. The mouse G was αq pTT plasmids encoding for Neuropilin-1 and VEGF (1:1 ratio, expressed at 16 mg/l, compared with a barely detectable level lane 1), Tie2 (lane 2), Cripto (lane 3) and c-Met (lane 4). All (by Commassie staining) when expressed from the pcDNA3.1 cultures were harvested 5 days post-transfection. With the vector (lane 4). exception of Cripto, which has been reported to be highly glycosylated on serine, threonine and asparagine (22), glyco- DISCUSSION sylation of the expressed proteins appeared to be relatively homogeneous as suggested by their migration behavior In this study, we investigated the effects of various parameters following SDS–PAGE. High expression levels of intracellular on r-protein expression by transient transfection of suspension PAGE 7 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 growing cells using the polycationic polymer PEI. By combining the use of the optimized oriP-containing pTT expression plasmid with the 293E cell line, we reached expression levels of intra- cellular r-protein representing up to 20% of total cellular proteins. To our knowledge, such high expression levels have never been described in 293 cells using transient transfection, and rival those obtained using virus-mediated transgene expression (12). Expression of the secreted protein SEAP was also considerable, as it was produced at levels exceeding 20 mg/l. The use of amplifiable expression cassettes in mammalian cells such as the dihydrofolate reductase or glutamine synthetase systems have been shown to result in the isolation of stable cell lines showing very high levels of r-protein expression. As an alternative to these stable amplified systems, vectors with viral-derived elements that allow for episomal replication and amplification, such as the large-T antigen/SV40 ori, or the EBNA1/oriP, are well suited when using transient expression systems (8). Although plasmid DNA containing the SV40 ori was shown to replicate in the large-T antigen expressing 293T cells line (23), we showed that it did not provide higher transgene expression in 293T cells when compared with the 293 parental cell line. In contrast, the use of oriP-containing plasmids in 293E cells significantly increased transgene expression compared with the non-permissive 293 cells. This suggests that the increased transgene expression obtained using EBV replicon-containing plasmids might be mediated by a phenomenon distinct from its ability to support episomal replication. This is further supported by the fact that removal of the DS domain of oriP, which is responsible for initiation of DNA replication in EBNA1 positive cells (24), did not significantly reduce transgene expression (data not shown). One likely Figure 7. SEAP purification and production of other secreted and intracellular r-proteins. (A) SEAP purification by IMAC. One liter of culture medium from mechanism for this oriP-mediated increased expression could the 3.5-l bioreactor harvest (Fig. 6) was loaded onto a TALON™ IMAC arise from the described EBNA1-dependent enhancer activity column (10 ml bed volume). Following extensive washing, bound material was of oriP (25–27). The EBV oriP contains 24 EBNA1 binding eluted with 150 mM imidazole (20 ml). Ten microliters of culture medium sites (28). As EBNA1 has an efficient nuclear localization (lane 1), flow-through (lane 2) and eluted material (lane 3) were resolved in signal (29,30), its binding to plasmids bearing an oriP may also duplicate on a 3–8% NuPAGE Tris–acetate gradient gel. One half of the gel was directly stained with Coomassie blue R-250 (left panel) whereas the other increase their nuclear import, thus enhancing transgene expression. half was transferred onto a nitrocellulose membrane and probed with anti-Myc Indeed, the most important barrier to transfection seems to be antibody (right panel). (B) Expression of secreted C-terminal Myc-(His) -tagged the limited migration of plasmid DNA from the cytoplasm to r-proteins in a 14-l bioreactor. Lane 1, human Neuropilin-1 (1–824; upper the nucleus (31). However, contribution of this mechanism to band) and VEGF (1–165; lower band) co-transfection in a 1:1 ratio; lane 2, human Tie2 (1–723); lane 3, human Cripto (1–173); lane 4, human c-Met (1–931). the enhanced transgene expression could be partially hindered Transfections were performed as described in Materials and Methods and cul- when using PEI as the transfection reagent, as this polymer ture medium harvested 120 hpt. Fifteen microliters of culture medium was was also shown to actively undergo nuclear localization loaded per lane and tagged proteins detected using anti-Myc antibody. (32,33). (C) Expression of intracellular r-proteins. Lane 1, pTT/sgGFP; lane 2, pTT/RR1; lane 3, pTT empty vector; lane 4, pcDNA3.1/G ; lane 5, pTT/G ; lane 6, Whereas linear 25 kDa PEI was reported to efficiently αq αq Kip1 19K pTT/p27 ; lane 7, pTT/PYC; lane 8, pTT/E1B ; lane 9, pTT/hexoki- mediate gene transfer in the presence of serum (34), transgene nase; lane 10, pTT/glucokinase. Cells were harvested 72 hpt, rinsed with PBS expression mediated by the branched isoform was shown to be and solubilized in NuPAGE sample buffer followed by sonication (lanes 1–5) reduced by 3-fold in its presence (6). This contrasts with our or extracted in lysis buffer (lanes 6–10) as indicated in Materials and Methods. results showing that gene transfer was also significantly Proteins were resolved on a 4–12% Bis–Tris NuPAGE gradient gel and stained with Coomassie blue R-250. increased using the branched 25 kDa PEI. The mechanism by which serum increases gene delivery and/or transgene expression is not yet clear. Serum might contribute to augment transcriptional A major drawback of gene transfer using polycations or cationic activity of the promoter as the CMV immediate early enhancer lipids is the inhibitory effect of conditioned medium on gene contains multiple binding sites for serum-activated transcription delivery. In the case of cationic lipids, this inhibition was factors (35,36). However, only a partial recovery of transgene shown to be mediated by the presence of secreted expression was obtained when serum was added to the cells 3 h glycosaminoglycans (21,37), which are expected to efficiently after their transfection in serum-free medium. This suggests displace DNA from lipid complexes. Whereas it was shown that, in addition to the potential serum-mediated CMV that conditioned medium adversely reduced PEI-mediated promoter transcriptional activation, some serum component(s) might increase transfection efficacy of DNA–PEI complexes. transfection of 293E cells (6), no significant effect was e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 8 OF 9 Table 1. Summary of r-protein expression levels –1 r-Protein Tag Localization Culture mode Concentration (mg l ) Human SEAP Myc-(His) Secreted 3-l bioreactor 20 Human Neuropilin-1 Myc-(His) Secreted 14-l bioreactor 8 Human VEGF Myc-(His) Secreted 14-l bioreactor 10 Human Tie2 Myc-(His) Secreted 3-l bioreactor 9 Human Cripto Myc-(His) Secreted 14-l bioreactor 9 Human c-Met Myc-(His) Secreted 14-l bioreactor 1 sgGFP None Intracellular Shaker flask 20 Herpes virus RR1 None Intracellular Shaker flask 50 Mouse Gα None Membrane T-flask 16 Kip1 Human p27 None Intracellular T-flask 14 Human hexokinase None Intracellular Shaker flask 40 Human glucokinase None Intracellular Shaker flask 30 Yeast PYC None Intracellular 1-l bioreactor 4 19K Adenovirus E1B None Intracellular T-flask 3 After purification by IMAC. Neuropilin-1 and VEGF were co-transfected. 3. Cachianes,G., Ho,C., Weber,R.F., Williams,S.R., Goeddel,D.V. and observed in our study. The reason for this discrepancy is not Leung,D.W. (1993) Epstein–Barr virus-derived vectors for transient and clear, but might result from the type of culture medium used, stable expression of recombinant proteins. Biotechniques, 15, 255–259. the age of the culture or from the cells themselves. The fact 4. Boussif,O., Lezoualc’h,F., Zanta,M.A., Mergny,M.D., Scherman,D., that, in our hands, transfection of cells in their 24 h-conditioned Demeneix,B. and Behr,J.P. (1995) A versatile vector for gene and medium does not reduce gene transfer and expression, greatly oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA, 92, 7297–7301. simplifies process scale-up. 5. Jordan,M., Kohne,C. and Wurm,F.M. (1998) Calcium-phosphate In conclusion, a significant improvement in transgene mediated DNA transfer into HEK-293 cells in suspension: control of expression following transient transfection of suspension- physicochemical parameters allows transfection in stirred media. growing cells using PEI was obtained by combining optimized Cytotechnology, 26, 39–47. 6. Schlaeger,E.-J. and Christensen,K. (1999) Transient gene expression in parameters such as the pTT expression vector, the 293E cell mammalian cells grown in serum-free suspension culture. line, the culture medium and the transfection process. Under Cytotechnology, 30, 71–83. these conditions, ∼60 mg of purified SEAP could be obtained 7. Wurm,F. and Bernard,A. (1999) Large-scale transient expression in from a 3-l culture following a single IMAC purification step. mammalian cells for recombinant protein production. Curr. Opin. Volumetric expressions of the intracellular proteins GFP and Biotechnol., 10, 156–159. 8. Van Craenenbroeck,K., Vanhoenacker,P. and Haegeman,G. (2000) RR1 were, respectively, 20 and 50 mg/l at 72 hpt, representing Episomal vectors for gene expression in mammalian cells. up to 20% of TCP. As this technology is robust, inexpensive Eur. J. Biochem., 267, 5665–5678. and easy to perform, it is fully adapted for high-throughput 9. Foecking,M.K. and Hofstetter,H. (1986) Powerful and versatile enhancer- production of milligram quantities of r-proteins needed for promoter unit for mammalian expression vectors. Gene, 45, 101–105. biochemical or structural studies and high-throughput screenings. 10. Gorman,C.M., Gies,D., McCray,G. and Huang,M. (1989) The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology, 171, 377–385. ACKNOWLEDGEMENTS 11. Massie,B., Couture,F., Lamoureux,L., Mosser,D.D., Guilbault,C., Jolicoeur,P., Belanger,F. and Langelier,Y. (1998) Inducible We thank Chunlin Xin, Eric Carpentier, Eric Thibaudeau and overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J. Virol., 72, 2289–2296. Robert Larocque for their technical assistance in cDNA 12. Massie,B., Mosser,D., Koutromanis,M., Vitté-Mony,I., Lamoureux,L., cloning and plasmid purification, Brian Cass for bioreactor Couture,F., Paquet,L., Guilbault,C., Dionne,J., Chahla,D. et al. (1998) runs, and Phuong Lan Pham, Gilles St-Laurent and Cynthia New adenovirus vectors for protein production and gene transfer. Elias for critical reading of the manuscript. We are grateful to Cytotechnology, 28, 53–64. Bernard Massie for providing us with purified RR1, anti-RR1 13. Godbey,W.T., Wu,K.K. and Mikos,A.G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Control Release, 60, 149–160. antibody and RR1 cDNA. 14. Côté,J., Garnier,A., Massie,B. and Kamen,A. (1998) Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol. Bioeng., 59, 567–575. REFERENCES 15. DuBridge,R.B., Tang,P., Hsia,H.C., Leong,P.M., Miller,J.H. and 1. Cullen,B.R. (1987) Use of eukaryotic expression technology in the Calos,M.P. (1987) Analysis of mutation in human cells by using an functional analysis of cloned genes. Methods Enzymol., 152, 684–704. Epstein–Barr virus shuttle system. Mol. Cell. Biol., 7, 379–387. 2. Blasey,H.D., Aubry,J.-P., Mazzei,G. and Bernard,A. (1996) Large scale 16. Kamen,A.A., Chavarie,C., André,J. and Archambault,J. (1992) Design transient expression with COS cells. Cytotechnology, 18, 183–192. parameters and performance of a surface baffled helical ribbon impeller PAGE 9 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 bioreactor for the culture of shear sensitive cells. Chem. Eng. Sci., 27, a viral protein required for viral DNA replication during latent infection. 2375–2380. J. Virol., 63, 2644–2649. 27. Gahn,T.A. and Sugden,B. (1995) An EBNA-1-dependent enhancer acts 17. Durocher,Y., Perret,S., Thibaudeau,E., Gaumond,M.H., Kamen,A., from a distance of 10 kilobase pairs to increase expression of the Stocco,R. and Abramovitz,M. (2000) A reporter gene assay for high- Epstein–Barr virus LMP gene. J. Virol., 69, 2633–2636. throughput screening of G-protein-coupled receptors stably or transiently 28. Mackey,D. and Sugden,B. (1999) Applications of oriP plasmids and their expressed in HEK293 EBNA cells grown in suspension culture. mode of replication. Methods Enzymol., 306, 308–328. Anal. Biochem., 284, 316–326. 29. Ambinder,R.F., Mullen,M.A., Chang,Y.N., Hayward,G.S. and 18. Chittenden,T., Frey,A. and Levine,A.J. (1991) Regulated replication of an Hayward,S.D. (1991) Functional domains of Epstein–Barr virus nuclear episomal simian virus 40 origin plasmid in COS7 cells. J. Virol., 65, antigen EBNA-1. J. Virol., 65, 1466–1478. 5944–5951. 30. Längle-Rouault,F., Patzel,V., Benavente,A., Taillez,M., Silvestre,N., 19. Yates,J.L., Warren,N. and Sugden,B. (1985) Stable replication of Bompard,A., Sczakiel,G., Jacobs,E. and Rittner,K. (1998) Up to 100-fold plasmids derived from Epstein–Barr virus in various mammalian cells. increase of apparent gene expression in the presence of Epstein–Barr virus Nature, 313, 812–815. oriP sequences and EBNA1: implications of the nuclear import of 20. Massie,B., Dionne,J., Lamarche,N., Fleurent,J. and Langelier,Y. (1995) plasmids. J. Virol., 72, 6181–6185. Improved adenovirus vector provides herpes simplex virus ribonucleotide 31. Zabner,J., Fasbender,A.J., Moninger,T., Poellinger,K.A. and Welsh,M.J. reductase R1 and R2 subunits very efficiently. Biotechnology, 13, 602–608. (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. 21. Ruponen,M., Yla-Herttuala,S. and Urtti,A. (1999) Interactions of J. Biol. Chem., 270, 18997–19007. polymeric and liposomal gene delivery systems with extracellular 32. Pollard,H., Remy,J.S., Loussouarn,G., Demolombe,S., Behr,J.P. and glycosaminoglycans: physicochemical and transfection studies. Escande,D. (1998) Polyethylenimine but not cationic lipids promotes Biochim. Biophys. Acta, 1415, 331–341. transgene delivery to the nucleus in mammalian cells. J. Biol. Chem., 273, 22. Schiffer,S.G., Foley,S., Kaffashan,A., Hronowski,X., Zichittella,A.E., 7507–7511. Yeo,C.Y., Miatkowski,K., Adkins,H.B., Damon,B., Whitman,M. et al. 33. Godbey,W.T., Wu,K.K. and Mikos,A.G. (1999) Tracking the intracellular (2001) Fucosylation of Cripto is required for its ability to facilitate nodal path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl signaling. J. Biol. Chem., 276, 37769–37778. Acad. Sci. USA, 96, 5177–5181. 23. Heinzel,S.S., Krysan,P.J., Calos,M.P. and DuBridge,R.B. (1988) Use of 34. Boussif,O., Zanta,M.A. and Behr,J.P. (1996) Optimized galenics improve simian virus 40 replication to amplify Epstein–Barr virus shuttle vectors in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther., in human cells. J. Virol., 62, 3738–3746. 3, 1074–1080. 24. Wysokenski,D.A. and Yates,J.L. (1989) Multiple EBNA1-binding sites 35. Boshart,M., Weber,F., Jahn,G., Dorsch-Hasler,K., Fleckenstein,B. and are required to form an EBNA1-dependent enhancer and to activate a Schaffner,W. (1985) A very strong enhancer is located upstream of an minimal replicative origin within oriP of Epstein–Barr virus. J. Virol., 63, immediate early gene of human cytomegalovirus. Cell, 41, 521–530. 2657–2666. 36. Brightwell,G., Poirier,V., Cole,E., Ivins,S. and Brown,K.W. (1997) 25. Reisman,D. and Sugden,B. (1986) trans-Activation of an Epstein–Barr Serum-dependent and cell cycle-dependent expression from a viral transcriptional enhancer by the Epstein–Barr viral nuclear antigen 1. cytomegalovirus-based mammalian expression vector. Gene, 194, 115–123. Mol. Cell. Biol., 6, 3838–3846. 37. Belting,M. and Petersson,P. (1999) Intracellular accumulation of secreted 26. Sugden,B. and Warren,N. (1989) A promoter of Epstein–Barr virus that proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer can function during latent infection can be transactivated by EBNA-1, of glycosaminoglycans to the nucleus. J. Biol. Chem., 274, 19375–19382. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

Loading next page...
 
/lp/oxford-university-press/high-level-and-high-throughput-recombinant-protein-production-by-UhvN4UgZIq

References (38)

Publisher
Oxford University Press
ISSN
0305-1048
eISSN
1362-4962
DOI
10.1093/nar/30.2.e9
Publisher site
See Article on Publisher Site

Abstract

© 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells Yves Durocher*, Sylvie Perret and Amine Kamen Animal Cell Technology and Downstream Processing Group, Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada Received July 16, 2001; Revised and Accepted November 11, 2001 ABSTRACT transient transfection of mammalian cells grown in monolayers can generate significant amounts of r-proteins (1–3) but scala- A scalable transfection procedure using polyethylen- bility of this process is limited by culture surface availability. imine (PEI) is described for the human embryonic The well established calcium phosphate precipitation tech- kidney 293 cell line grown in suspension. Green nique or the recently described cationic polymer polyethylen- fluorescent protein (GFP) and human placental imine (PEI) (4) provides cost effective vehicles for the secreted alkaline phosphatase (SEAP) were used as introduction of plasmid DNA into mammalian cells. A major reporter genes to monitor transfection efficiency and breakthrough has recently emerged for the fast production of productivity. Up to 75% of GFP-positive cells were milligram amounts of recombinant proteins when these gene transfer vehicles were shown to be effective for the large-scale obtained using linear or branched 25 kDa PEI. The transfection of mammalian cells grown in suspension culture 293 cell line and two genetic variants, either (5–7). expressing the SV40 large T-antigen (293T) or the For an optimal large-scale transient transfection and r-protein Epstein–Barr virus (EBV) EBNA1 protein (293E), were expression in mammalian cells, four key aspects need to be tested for protein expression. The highest expression taken into account, namely (i) the cell line, (ii) the expression level was obtained with 293E cells using the EBV vector, (iii) the transfection vehicle and (iv) the culture oriP-containing plasmid pCEP4. We designed the medium. The human 293 cell line is widely used for r-protein pTT vector, an oriP-based vector having an improved production as it offers many advantages such as high transfection cytomegalovirus expression cassette. Using this yields with most gene transfer vehicles, is easily grown in vector, 10- and 3-fold increases in SEAP expression suspension culture, and can be adapted to serum-free media. was obtained in 293E cells compared with pcDNA3.1 Moreover, two genetic variants, the 293E and 293T cell lines, and pCEP4 vectors, respectively. The presence of expressing the Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen, allow episomal ampli- serum had a positive effect on gene transfer and fication of plasmids containing the viral EBV (293E) or SV40 expression. Transfection of suspension-growing (293T) origins of replication. Thus, they are expected to cells was more efficient with linear PEI and was not increase r-protein expression levels by permitting more affected by the presence of medium conditioned for plasmid copies to persist in the transfected cells throughout the 24 h. Using the pTT vector, >20 mg/l of purified His- production phase (8). The second important issue for high level tagged SEAP was recovered from a 3.5 l bioreactor. r-protein expression is to use vectors with promoters that are Intracellular proteins were also produced at levels as highly active in the host cell line, such as the human cyto- high as 50 mg/l, representing up to 20% of total cell megalovirus (CMV) promoter (9). This promoter is particularly proteins. powerful in 293 cells where it has been shown to be strongly transactivated by the constitutively expressed adenovirus E1a protein (10). Moreover, a highly efficient expression cassette INTRODUCTION using this promoter was recently described that provides Mammalian cells are an established expression system in the adenovirus-mediated transgene expression levels reaching up biotechnology industry for the production of recombinant to 20% of total cell proteins (TCP) (11,12). The third aspect is proteins (r-proteins). In contrast to lower eukaryotes or related to gene transfer reagent efficacy. Even though many prokaryotes, mammalian cells provide active r-proteins that highly effective gene transfer reagents are commercially possess relevant post-translational modifications. However, in available, only a few are cost effective when considering order to obtain sufficient amounts of protein for structure/activity operations at the multi-liters scale. For large-scale transient analyses or high-throughput screenings, one needs to go transfection applications, these reagents should also be simple through the long and tedious process of stable transfectoma to use, effective with suspension-growing cells and have isolation and characterization. As an alternative, the small-scale minimal cytotoxic effects. PEI satisfies most of these criteria *To whom correspondence should be addressed. Tel: +1 514 496 6192; Fax: +1 514 496 6785; Email: yves.durocher@nrc.ca e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 2 OF 9 as it has high gene transfer activity in many cell lines while pCEP5 vector. The pTT vector was generated following displaying low cytotoxicity (4), is cost effective and efficiently deletion of the hygromycin (BsmI and SalI excision followed transfects suspension growing 293 cells (6). This polymer is by fill-in and ligation) and EBNA1 (ClaI and NsiI excision available as both linear and branched isoforms with a wide followed by fill-in and ligation) expression cassettes. The ColE1 range of molecular weights and polydispersities, these physico- origin (FspI–SalI fragment, including the 3′ end of β-lactamase chemical parameters being critical for efficient gene transfer ORF) was replaced with a FspI–SalI fragment from pcDNA3.1 activity (13). The last key aspect for efficient protein expression containing the pMB1 origin (and the same 3′ end of β-lactamase by transient transfection deals with the culture medium. Some ORF). A Myc-(His) C-terminal fusion tag was added to SEAP gene transfer reagents work only in serum-free media whereas (HindIII–HpaI fragment from pSEAP-basic) following in-frame others are insensitive to the presence of serum. Also, as the ligation in pcDNA3.1/Myc-His digested with HindIII and presence of cellular by-products in conditioned medium is EcoRV. All plasmids were amplified in Escherichia coli (DH5α) associated with poor transfection yield, it is often necessary to grown in LB medium and purified using MAXI prep columns perform a complete medium exchange prior to transfection. (Qiagen, Mississauga, Ontario, Canada). For quantification, However, this step does not satisfy the need for a robust large- plasmids were diluted in 50 mM Tris–HCl pH 7.4 and the scale transient transfection process. absorbances at 260 and 280 nm measured. Only plasmid preparations with A /A ratios between 1.75 and 2.00 were In this study, the model proteins, green fluorescent protein 260 280 used. (GFP) and secreted alkaline phosphatase (SEAP), were used to design an expression vector and establish transfection para- Small-scale transient transfections meters in order to reach high expression levels in suspension growing 293E cells using both linear and branched 25 kDa Three hours before transfection, cells were centrifuged and PEI. We also show that this technology is fully adapted for the resuspended in fresh HSFM supplemented with 1% BCS at a 6 –1 high-throughput production of r-proteins and will assuredly be density of 1.0 × 10 cells ml . Five hundred microliters, or useful for structure–function studies and high-throughput 10 ml, of cell suspension was distributed per well in a 12-well screening assays. plate, or in a 125 ml shaker flask, respectively. DNA was diluted in fresh serum-free HSFM (in a volume equivalent to one-tenth of the culture to be transfected), PEI was added, and MATERIALS AND METHODS the mixture immediately vortexed and incubated for 10 min at room temperature prior to its addition to the cells. Following a Chemicals 3 h incubation with DNA–PEI complexes, culture medium was A 25 kDa branched PEI was obtained from Aldrich completed to 1 ml (12-well plate) or 20 ml (shaker flask) by the (Milwaukee, WI) and 25 kDa linear PEI from Polysciences addition of HSFM supplemented with 1% BCS. –1 (Warrington, PA). Stock solutions (1 mg ml ) were prepared Transfection in bioreactors in water, neutralized with HCl, sterilized by filtration (0.22 µ m), aliquoted and stored at –80°C. A 3.5-l bioreactor containing 2.85 l of HSFM supplemented with 1% BCS was seeded with 293E cells to obtain a final cell Cell culture 5 –1 density of 2.5 × 10 ml . Twenty-four hours later, cells were Human embryonic kidney 293S (293) cells (14) and genetic transfected with 150 ml of a mixture of pTT/SEAP:pEGFP variants stably expressing EBNA1 (293E) (Invitrogen, plasmids (19:1, 3 mg total) and PEI (6 mg). Agitation was at Carlsbad, CA) or the large-T antigen (293T) (15) were adapted 70 r.p.m. using a helical ribbon impeller (16). Dissolved to suspension culture in low-calcium-hybridoma serum-free oxygen was maintained at 40% by surface aeration using a medium (HSFM) (14) supplemented with 1% bovine calf serum nitrogen/oxygen mixture (300 ml/min) and pH was maintained –1 (BCS), 50 µ g ml Geneticin (for 293E and 293T cells), 0.1% at 7.2 by addition of CO in the head space and sodium Pluronic F-68 (Sigma, Oakville, Ontario, Canada) and 10 mM bicarbonate [10% (w/v) in water] injection in the culture HEPES. For culture in bioreactors, HEPES was omitted from the medium. The same conditions were used for transfection in 14-l medium. Cells were cultured in Erlenmeyer flasks (50 or 125 ml) bioreactors. using 15–25% of the nominal volume at 110–130 r.p.m. Flow cytometry (Thermolyne’s BigBill orbital shaker; TekniScience Inc., Terre- bonne, Québec, Canada) under standard humidified conditions GFP was analyzed by flow cytometry using an EPICS Profile (37°C and 5% CO ). II (Coulter, Hialeah, FL) equipped with a 15-mW argon-ion laser. Only viable cells were analyzed for the expression of Vectors GFP. Data are representative of at least two independent The pIRESpuro/EGFP (pEGFP) and pSEAP basic vectors experiments. Error bars represent ±SEM of one experiment were obtained from Clontech (Palo Alto, CA), and pcDNA3.1, done in duplicate. pcDNA3.1/Myc-(His) and pCEP4 vectors were from Invitrogen. SEAP analysis The SuperGlo GFP variant (sgGFP) was from Q·Biogene (Carlsbad, CA). Construction of pCEP5 vector was as follows: Determination of SEAP activity was performed essentially as the CMV promoter and polyadenylation signal of pCEP4 were previously described by Durocher et al. (17). Briefly, culture removed by sequential digestion and self-ligation using SalI medium was diluted in water as required (typically 1/50 to 1/1000) and XbaI enzymes, resulting in plasmid pCEP4∆ . A BglII fragment and 50 µ l was transferred to a 96-well plate. Fifty microliters from pAdCMV5 (11) encoding the CMV5-poly(A) expression of SEAP assay solution containing 20 mM paranitrophenyl- cassette was ligated in BglII-linearized pCEP4∆ , resulting in phosphate (pNPP), 1 mM MgCl , 10 mM l-homoarginine and 2 PAGE 3 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 1 M diethanolamine pH 9.8 were then added and absorbance read at 410 nm at 1–2 min intervals at room temperature to determine pNPP hydrolysis rates. Data are representative of at least two independent experiments. Error bars represent ±SEM of one experiment done in duplicate. For the bioreactor run, error bars represent ±SEM of two SEAP measurements. Electrophoresis, western analyses and quantification Immunodetection of C-terminal Myc-(His) -tagged SEAP was done using the anti-Myc 9E10 antibody (Santa Cruz). For analysis of intracellular proteins, cells were solubilized in NuPAGE sample buffer (Novex) or extracted with lysis buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 1% Thesit and 0.5% sodium deoxycholate). Insoluble material was removed from lysates by centrifugation at 12 000 g at 4°C for 5 min. Concentrated NuPAGE buffer (4×) was added to cleared lysates. All samples were heated for 3 min at 95°C. Proteins were resolved on 4–12% Bis–Tris or 3–8% Tris–acetate NuPAGE gradient gels as recommended by the manufacturer. GFP and other non-tagged proteins were quantified relative to purified bovine serum albumin (BSA) following electrophoresis and Coomassie blue R250 staining using the Kodak Digital Science Image Station 440cf equipped with the Kodak Digital Science 1D image analysis software version 3.0 (Eastman Kodak, NY). RR1 was quantified by slot-blot relative to a homogeneity-purified RR1 standard detected by using a monoclonal anti-RR1 antibody. Other Myc-(His) -tagged proteins were quantified relative to purified SEAP-Myc-(His) . RESULTS Figure 1. Effect of DNA to PEI ratio on transfection efficiency. 293E cells were Transfection with linear and branched 25 kDa PEI transfected with linear (A) or branched (B) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations Preliminary results showed that linear and branched 25 kDa –1 (µ g ml ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 PEI were the most effective among various polymers tested (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt. (including branched 70 kDa, branched 50–100 kDa and branched 10 kDa; data not shown). Therefore, we optimized transfection of 293E cells with both linear or branched 25 kDa plasmid DNA up to 90 copies in cells expressing the EBNA1 PEI polymers using a plasmid encoding the enhanced GFP protein (19). We also generated the pCEP5 vector (Fig. 2A, (pEGFP). Transfections were performed using cells grown as left) by using an improved CMV expression cassette as monolayers in 12-well plates and GFP expression was measured described in the adenoviral transfer vector pAdCMV5 (20). 72 h later by flow cytometry. The effect of DNA to PEI ratios This expression cassette has been shown to confer very high on transfection efficiency is shown in Figure 1 using linear (A) levels of r-protein expression in 293 cells (12). The pCEP5 or branched (B) PEI. The indicated amounts of DNA and vector was further modified (Materials and Methods) to yield polymer are for one well containing 5 × 10 cells. Only 0.25 µ g the pTT vector (Fig. 2A, right) that is 4.6 kb smaller, hence of DNA was sufficient to reach a 50% transfection efficiency using linear PEI, whereas a minimum of 1.0 µ g was necessary providing more space for large cDNA cloning. The cDNA using the branched isoform. Transfection efficiencies of ∼70% encoding for the reporter protein SEAP was then cloned in were reached with both linear and branched polymers at each of these four vectors and its expression level monitored DNA:PEI (µ g:µ g) ratios of 1.0:1.5 and 1.5:2.0, respectively. following transient transfection in 293, 293T or 293E cells. As Increasing the amounts of both DNA and PEI did not lead to shown in Figure 2B, transfection of the 293T cell line with the higher transfection yield. SV40 ori-containing plasmid pcDNA3.1 did not translate into an increased transgene expression when compared with trans- Cell line and expression vectors fection of the parental 293 cells. However, transfection of 293E cells with the pCEP4 vector resulted in a 2–3-fold Two commercially available expression vectors containing increase in SEAP expression compared with transfection of 293 viral sequences allowing for episomal DNA replication in or 293T cells with the same vector. In addition, the use of the permissive cell lines were tested. The first vector, pcDNA3.1, contains the SV40 origin of replication that allows cellular pCEP5 vector further increased SEAP expression by a factor polymerases to replicate the DNA up to 10 000 copies in cells of 2–6-fold, depending on the cell line. Finally, the use of the expressing the large T antigen (18). The second vector, pCEP4, pTT vector in 293E cells resulted in a 33% increase in trans- contains the EBV origin of replication oriP that replicates gene expression compared with the pCEP5 vector. The overall e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 4 OF 9 Figure 2. Effect of cell lines and vectors on SEAP expression. (A) Genetic maps of pCEP5 (left) and pTT (right) vectors drawn to scale. The pCEP5 vector backbone is identical to pCEP4 vector except for the transgene expression cassette. Construction of the pTT vector is as described in Materials and Methods. TPL, tripartite leader; enh MLP, adenovirus major late promoter enhancer; SD, splice donor; SA, splice acceptor; DS, dyad symmetry; FR, family of repeats. (B) Cells were transfected with 1 µ g of DNA and 2 µ g of linear PEI and SEAP activity measured 72 hpt. The pEGFP plasmid (0.1 µ g) was also added in each condition to monitor for transfection efficiency and SEAP activities were normalized accordingly. Open boxes, pcDNA3.1/SEAP; hatched boxes, pCEP4/SEAP; gray boxes, pCEP5/SEAP; closed boxes, pTT/SEAP. SEAP expression level in 293E cells was 10-fold higher with BCS-supplemented HSFM. Shaker flask cultures were co-trans- the pTT vector compared with the pcDNA3.1 vector. fected with a mixture of pTT/SEAP:pEGFP (9:1) plasmids (pEGFP was added to monitor transfection efficiency). With Effect of serum both linear and branched PEI, SEAP accumulated in the culture medium for up to 96 hours post-transfection (hpt) (Fig. 4), but The effect of serum on transfection efficiency (GFP) and r-protein gene transfer and expression level were 50% higher using the production (SEAP) mediated by both linear and branched PEI was evaluated. Figure 3 shows that when transfection mixture was linear isoform. These results clearly demonstrate that linear, and to a lesser extent branched, PEI are effective for gene transfer added to cells in fresh 1% serum-containing medium, a 4–5-fold in suspension-growing cells. In addition, SEAP expression increase in SEAP activity was obtained compared with its levels obtained with suspension-growing cells using linear PEI addition to cells in serum-free medium. Increasing serum concentration up to 5% further improved PEI-mediated trans- were comparable with those obtained with adherent-growing fection efficiency and production. When transfection mixture cells. For all experiments reported below, only linear PEI was was added to cells in serum-free media followed 3 h later by used. serum addition to a concentration of 1% (0→1%), a 2-fold Our goal was to define a robust, simple and scalable trans- increase in transgene expression was obtained; however, this fection process. In order to reach these objectives, two steps level was only 50% of that obtained in 1% serum. had to be simplified: the 3 h incubation of DNA–PEI complexes with cells in a reduced culture volume, and the Process optimization for transfection in suspension medium change 3 h prior to transfection. The first step was We next evaluated gene transfer efficiency of both linear and performed with the assumption that it would promote inter- branched PEI on suspension-growing 293E cells in 1% action of the DNA–PEI complexes with the cells and thus PAGE 5 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 Figure 4. Transfection of suspension growing cells. Cells were resuspended in 6 –1 10 ml of fresh HSFM containing 1% BCS to a density of 1 × 10 ml in a 125 ml Erlenmeyer flask. Three hours later, 1 ml of the DNA–PEI complexes were added and the culture incubated for an additional 3 h. The volume was then completed to 20 ml with fresh culture medium. The DNA–PEI complexes were as follows: 40 µ g of linear or branched PEI was added to 1 ml of HEPES- supplemented HSFM containing 18 µ g of pTT/SEAP and 2 µ g of pEGFP or 27 µ g of pTT/SEAP and 3 µ g of pEGFP, respectively. Open symbols, linear PEI; closed symbols: branched PEI. observed when the transfection was carried out in medium conditioned for 24 h, indicating that medium exchange is not necessary. Transfection in bioreactors To demonstrate scalability of the process, a 3.5-l bioreactor Figure 3. Effect of serum on transgene expression. 293E cells were transfected culture was transfected with a mixture of pTT/SEAP:pEGFP with pTT/sgGFP (A) or pTT/SEAP (B) vectors using 1.0 µ g of DNA and plasmids (19:1). One hour later, a sample (25 ml) was withdrawn 2.0 µ g of linear PEI (hatched boxes) or 1.5 and 2.0 µ g of branched PEI (gray and transferred into a shaker flask as a control. In the bioreactor boxes) in fresh serum-free or serum-supplemented media. In one experiment (0→1%), cells were transfected in serum-free media and serum was added 3 h (Fig. 6A, unbroken lines), SEAP (circles) accumulated up to later to a final concentration of 1%. GFP-positive cells and SEAP activity were 144 hpt and then reached a plateau whereas accumulation measured 72 hpt. continued up to 216 hpt in the control shaker flask (dashed lines). The percentage of GFP-positive cells (squares) at 96 hpt reached 54 and 50% for the bioreactor and the shaker flask, increase transfection efficiency. The second was done according to reports showing a deleterious effect of conditioned medium on respectively. At the end of the culture, cell density was 4.1 and 6 –1 transfection efficiency (6,21). Whereas medium exchange is 4.7 × 10 ml with a viability of 62 and 72% for the bioreactor simple to perform on a small scale, this step represents a and shaker flask, respectively (Fig. 6B). Although viable cell significant hurdle at scales greater than a few liters. density was 25% lower in the bioreactor compared with the shaker flask, volumetric SEAP productivity was almost 2-fold The effect of cell density at time of transfection was first evaluated (Fig. 5A) by transfecting high density (hatched bars; higher. Similar results were systematically observed in five 6 –1 10 ml at 1 × 10 cells ml ) or low density cultures (gray bars; independent experiments (results not shown), indicating that 5 –1 the productivity of secreted proteins might be increased when 20 ml at 5 × 10 cells ml ) in shaker flasks. Three hours later, 5 –1 using a controlled environment. the high cell density flask was diluted to 5 × 10 cells ml with fresh medium, and GFP expression in both flasks monitored Purification of SEAP and production of other r-proteins 72 h later. This experiment showed that cell concentration prior to transfection could be omitted as only a slight decrease Purification of Myc-(His) -tagged SEAP harvested from the (<10%) in transfection efficiency and a 15% decrease in GFP bioreactor run (Fig. 6) by immobilized metal affinity chroma- expression level were observed when cells were transfected in tography (IMAC) is shown in Figure 7A. The left panel shows a larger culture volume. Coomassie blue-stained protein pattern from the culture We next evaluated the effect of conditioned medium on medium before loading on the column (lane 1), flow-through SEAP expression using suspension growing cells. For this (lane 2) and eluted material using 150 mM imidazole (lane 3). study, cells were seeded in shaker flasks at a density of 2.5 × The right panel shows immunodetection of SEAP in the same 5 –1 10 ml . Twenty-four hours later, transfection was performed fractions using anti-Myc antibody. This figure shows that all of with or without a complete medium exchange. As shown in the His-tagged SEAP was retained on the column whereas very Figure 5B, no significant difference in SEAP expression was few, if any, serum proteins bound to it (SEAP migrates with an e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 6 OF 9 Figure 6. Transient transfection in a 3.5-l bioreactor. (A) 293E cells were 5 –1 seeded at a density of 2.5 × 10 ml in 2.85 l of fresh HSFM supplemented with 1% BCS. Twenty-four hours later, the transfection mixture (6 mg of linear PEI added to 150 ml HSFM containing 2.85 mg pTT/SEAP and 150 µ g pEGFP plasmids) was added to the bioreactor (unbroken lines). One hour later, Figure 5. Effect of cell density and of conditioned medium. (A) Transfection 25 ml of culture was withdrawn from the bioreactor and transferred in a shake efficiency and relative total GFP expression (in percent) obtained following flask as a control (dashed lines). SEAP activity (circles) and GFP-positive cells 6 –1 transfection using standard conditions (hatched bars: 10 ml of cells at 1 × 10 ml (squares) were determined as described in Materials and Methods. (B) Growth followed by addition of 10 ml of fresh medium 3 h after transfection) or using curves (diamonds), viability (triangles) and yO (gray line) in the 3.5-l bioreactor 5 –1 cells at 5 × 10 ml in 20 ml of culture medium (gray bars). GFP was monitored (unbroken lines) and shaker flask (dashed lines). 72 hpt. Relative total GFP was obtained following multiplication of percent GFP-positive cells by the mean fluorescence intensity. (B) Cells were seeded 5 –1 in 20 ml of 1% BCS-supplemented HSFM at a density of 2.5 × 10 ml 24 h before transfection. The medium was then left unchanged (conditioned: open r-proteins were also obtained as shown in Figure 7C. In this circles) or replaced with 20 ml of fresh medium (closed circles). Three hours experiment, 293E cells were transfected with pTT plasmids later, cells were transfected by the addition of 2 ml of DNA–PEI complexes encoding for sgGFP (lane 1), herpes simplex virus ribonucleo- (20 µ g of pTT/SEAP and 40 µ g of linear PEI). tide reductase 1 (RR1, lane 2), mouse G (lane 5), human αq Kip1 p27 (lane 6), yeast pyruvate carboxylase (PYC, lane 7), 19K adenovirus E1B (lane 8), human hexokinase 1 (HK, lane 9) apparent molecular weight slightly higher than BSA). SEAP and human glucokinase (GK, lane 10). Three days after trans- quantification in the eluted fraction using the Lowry protein fection, cells were rinsed with PBS, solubilized in sample assay showed that ∼60 mg of His-tagged SEAP could be buffer (GFP, RR1 and G ) or extracted with lysis buffer αq recovered by IMAC from the 3-l bioreactor culture. As shown Kip1 19K (p27 , PYC, E1B , HK and GK), and proteins analyzed by in Figure 7B, high expression levels in bioreactor were also SDS–PAGE. Quantification of r-proteins shown in Figure 7 is obtained with other secreted r-proteins. Fourteen- (lanes 1, 3 summarized in Table 1. In the case of RR1, volumetric production and 4) or 3.5-liter (lane 2) bioreactors were transfected with was 50 mg/l, representing 20% of TCP. The mouse G was αq pTT plasmids encoding for Neuropilin-1 and VEGF (1:1 ratio, expressed at 16 mg/l, compared with a barely detectable level lane 1), Tie2 (lane 2), Cripto (lane 3) and c-Met (lane 4). All (by Commassie staining) when expressed from the pcDNA3.1 cultures were harvested 5 days post-transfection. With the vector (lane 4). exception of Cripto, which has been reported to be highly glycosylated on serine, threonine and asparagine (22), glyco- DISCUSSION sylation of the expressed proteins appeared to be relatively homogeneous as suggested by their migration behavior In this study, we investigated the effects of various parameters following SDS–PAGE. High expression levels of intracellular on r-protein expression by transient transfection of suspension PAGE 7 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 growing cells using the polycationic polymer PEI. By combining the use of the optimized oriP-containing pTT expression plasmid with the 293E cell line, we reached expression levels of intra- cellular r-protein representing up to 20% of total cellular proteins. To our knowledge, such high expression levels have never been described in 293 cells using transient transfection, and rival those obtained using virus-mediated transgene expression (12). Expression of the secreted protein SEAP was also considerable, as it was produced at levels exceeding 20 mg/l. The use of amplifiable expression cassettes in mammalian cells such as the dihydrofolate reductase or glutamine synthetase systems have been shown to result in the isolation of stable cell lines showing very high levels of r-protein expression. As an alternative to these stable amplified systems, vectors with viral-derived elements that allow for episomal replication and amplification, such as the large-T antigen/SV40 ori, or the EBNA1/oriP, are well suited when using transient expression systems (8). Although plasmid DNA containing the SV40 ori was shown to replicate in the large-T antigen expressing 293T cells line (23), we showed that it did not provide higher transgene expression in 293T cells when compared with the 293 parental cell line. In contrast, the use of oriP-containing plasmids in 293E cells significantly increased transgene expression compared with the non-permissive 293 cells. This suggests that the increased transgene expression obtained using EBV replicon-containing plasmids might be mediated by a phenomenon distinct from its ability to support episomal replication. This is further supported by the fact that removal of the DS domain of oriP, which is responsible for initiation of DNA replication in EBNA1 positive cells (24), did not significantly reduce transgene expression (data not shown). One likely Figure 7. SEAP purification and production of other secreted and intracellular r-proteins. (A) SEAP purification by IMAC. One liter of culture medium from mechanism for this oriP-mediated increased expression could the 3.5-l bioreactor harvest (Fig. 6) was loaded onto a TALON™ IMAC arise from the described EBNA1-dependent enhancer activity column (10 ml bed volume). Following extensive washing, bound material was of oriP (25–27). The EBV oriP contains 24 EBNA1 binding eluted with 150 mM imidazole (20 ml). Ten microliters of culture medium sites (28). As EBNA1 has an efficient nuclear localization (lane 1), flow-through (lane 2) and eluted material (lane 3) were resolved in signal (29,30), its binding to plasmids bearing an oriP may also duplicate on a 3–8% NuPAGE Tris–acetate gradient gel. One half of the gel was directly stained with Coomassie blue R-250 (left panel) whereas the other increase their nuclear import, thus enhancing transgene expression. half was transferred onto a nitrocellulose membrane and probed with anti-Myc Indeed, the most important barrier to transfection seems to be antibody (right panel). (B) Expression of secreted C-terminal Myc-(His) -tagged the limited migration of plasmid DNA from the cytoplasm to r-proteins in a 14-l bioreactor. Lane 1, human Neuropilin-1 (1–824; upper the nucleus (31). However, contribution of this mechanism to band) and VEGF (1–165; lower band) co-transfection in a 1:1 ratio; lane 2, human Tie2 (1–723); lane 3, human Cripto (1–173); lane 4, human c-Met (1–931). the enhanced transgene expression could be partially hindered Transfections were performed as described in Materials and Methods and cul- when using PEI as the transfection reagent, as this polymer ture medium harvested 120 hpt. Fifteen microliters of culture medium was was also shown to actively undergo nuclear localization loaded per lane and tagged proteins detected using anti-Myc antibody. (32,33). (C) Expression of intracellular r-proteins. Lane 1, pTT/sgGFP; lane 2, pTT/RR1; lane 3, pTT empty vector; lane 4, pcDNA3.1/G ; lane 5, pTT/G ; lane 6, Whereas linear 25 kDa PEI was reported to efficiently αq αq Kip1 19K pTT/p27 ; lane 7, pTT/PYC; lane 8, pTT/E1B ; lane 9, pTT/hexoki- mediate gene transfer in the presence of serum (34), transgene nase; lane 10, pTT/glucokinase. Cells were harvested 72 hpt, rinsed with PBS expression mediated by the branched isoform was shown to be and solubilized in NuPAGE sample buffer followed by sonication (lanes 1–5) reduced by 3-fold in its presence (6). This contrasts with our or extracted in lysis buffer (lanes 6–10) as indicated in Materials and Methods. results showing that gene transfer was also significantly Proteins were resolved on a 4–12% Bis–Tris NuPAGE gradient gel and stained with Coomassie blue R-250. increased using the branched 25 kDa PEI. The mechanism by which serum increases gene delivery and/or transgene expression is not yet clear. Serum might contribute to augment transcriptional A major drawback of gene transfer using polycations or cationic activity of the promoter as the CMV immediate early enhancer lipids is the inhibitory effect of conditioned medium on gene contains multiple binding sites for serum-activated transcription delivery. In the case of cationic lipids, this inhibition was factors (35,36). However, only a partial recovery of transgene shown to be mediated by the presence of secreted expression was obtained when serum was added to the cells 3 h glycosaminoglycans (21,37), which are expected to efficiently after their transfection in serum-free medium. This suggests displace DNA from lipid complexes. Whereas it was shown that, in addition to the potential serum-mediated CMV that conditioned medium adversely reduced PEI-mediated promoter transcriptional activation, some serum component(s) might increase transfection efficacy of DNA–PEI complexes. transfection of 293E cells (6), no significant effect was e9 Nucleic Acids Research, 2002, Vol. 30, No. 2 PAGE 8 OF 9 Table 1. Summary of r-protein expression levels –1 r-Protein Tag Localization Culture mode Concentration (mg l ) Human SEAP Myc-(His) Secreted 3-l bioreactor 20 Human Neuropilin-1 Myc-(His) Secreted 14-l bioreactor 8 Human VEGF Myc-(His) Secreted 14-l bioreactor 10 Human Tie2 Myc-(His) Secreted 3-l bioreactor 9 Human Cripto Myc-(His) Secreted 14-l bioreactor 9 Human c-Met Myc-(His) Secreted 14-l bioreactor 1 sgGFP None Intracellular Shaker flask 20 Herpes virus RR1 None Intracellular Shaker flask 50 Mouse Gα None Membrane T-flask 16 Kip1 Human p27 None Intracellular T-flask 14 Human hexokinase None Intracellular Shaker flask 40 Human glucokinase None Intracellular Shaker flask 30 Yeast PYC None Intracellular 1-l bioreactor 4 19K Adenovirus E1B None Intracellular T-flask 3 After purification by IMAC. Neuropilin-1 and VEGF were co-transfected. 3. Cachianes,G., Ho,C., Weber,R.F., Williams,S.R., Goeddel,D.V. and observed in our study. The reason for this discrepancy is not Leung,D.W. (1993) Epstein–Barr virus-derived vectors for transient and clear, but might result from the type of culture medium used, stable expression of recombinant proteins. Biotechniques, 15, 255–259. the age of the culture or from the cells themselves. The fact 4. Boussif,O., Lezoualc’h,F., Zanta,M.A., Mergny,M.D., Scherman,D., that, in our hands, transfection of cells in their 24 h-conditioned Demeneix,B. and Behr,J.P. (1995) A versatile vector for gene and medium does not reduce gene transfer and expression, greatly oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl Acad. Sci. USA, 92, 7297–7301. simplifies process scale-up. 5. Jordan,M., Kohne,C. and Wurm,F.M. (1998) Calcium-phosphate In conclusion, a significant improvement in transgene mediated DNA transfer into HEK-293 cells in suspension: control of expression following transient transfection of suspension- physicochemical parameters allows transfection in stirred media. growing cells using PEI was obtained by combining optimized Cytotechnology, 26, 39–47. 6. Schlaeger,E.-J. and Christensen,K. (1999) Transient gene expression in parameters such as the pTT expression vector, the 293E cell mammalian cells grown in serum-free suspension culture. line, the culture medium and the transfection process. Under Cytotechnology, 30, 71–83. these conditions, ∼60 mg of purified SEAP could be obtained 7. Wurm,F. and Bernard,A. (1999) Large-scale transient expression in from a 3-l culture following a single IMAC purification step. mammalian cells for recombinant protein production. Curr. Opin. Volumetric expressions of the intracellular proteins GFP and Biotechnol., 10, 156–159. 8. Van Craenenbroeck,K., Vanhoenacker,P. and Haegeman,G. (2000) RR1 were, respectively, 20 and 50 mg/l at 72 hpt, representing Episomal vectors for gene expression in mammalian cells. up to 20% of TCP. As this technology is robust, inexpensive Eur. J. Biochem., 267, 5665–5678. and easy to perform, it is fully adapted for high-throughput 9. Foecking,M.K. and Hofstetter,H. (1986) Powerful and versatile enhancer- production of milligram quantities of r-proteins needed for promoter unit for mammalian expression vectors. Gene, 45, 101–105. biochemical or structural studies and high-throughput screenings. 10. Gorman,C.M., Gies,D., McCray,G. and Huang,M. (1989) The human cytomegalovirus major immediate early promoter can be trans-activated by adenovirus early proteins. Virology, 171, 377–385. ACKNOWLEDGEMENTS 11. Massie,B., Couture,F., Lamoureux,L., Mosser,D.D., Guilbault,C., Jolicoeur,P., Belanger,F. and Langelier,Y. (1998) Inducible We thank Chunlin Xin, Eric Carpentier, Eric Thibaudeau and overexpression of a toxic protein by an adenovirus vector with a tetracycline-regulatable expression cassette. J. Virol., 72, 2289–2296. Robert Larocque for their technical assistance in cDNA 12. Massie,B., Mosser,D., Koutromanis,M., Vitté-Mony,I., Lamoureux,L., cloning and plasmid purification, Brian Cass for bioreactor Couture,F., Paquet,L., Guilbault,C., Dionne,J., Chahla,D. et al. (1998) runs, and Phuong Lan Pham, Gilles St-Laurent and Cynthia New adenovirus vectors for protein production and gene transfer. Elias for critical reading of the manuscript. We are grateful to Cytotechnology, 28, 53–64. Bernard Massie for providing us with purified RR1, anti-RR1 13. Godbey,W.T., Wu,K.K. and Mikos,A.G. (1999) Poly(ethylenimine) and its role in gene delivery. J. Control Release, 60, 149–160. antibody and RR1 cDNA. 14. Côté,J., Garnier,A., Massie,B. and Kamen,A. (1998) Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol. Bioeng., 59, 567–575. REFERENCES 15. DuBridge,R.B., Tang,P., Hsia,H.C., Leong,P.M., Miller,J.H. and 1. Cullen,B.R. (1987) Use of eukaryotic expression technology in the Calos,M.P. (1987) Analysis of mutation in human cells by using an functional analysis of cloned genes. Methods Enzymol., 152, 684–704. Epstein–Barr virus shuttle system. Mol. Cell. Biol., 7, 379–387. 2. Blasey,H.D., Aubry,J.-P., Mazzei,G. and Bernard,A. (1996) Large scale 16. Kamen,A.A., Chavarie,C., André,J. and Archambault,J. (1992) Design transient expression with COS cells. Cytotechnology, 18, 183–192. parameters and performance of a surface baffled helical ribbon impeller PAGE 9 OF 9 Nucleic Acids Research, 2002, Vol. 30, No. 2 e9 bioreactor for the culture of shear sensitive cells. Chem. Eng. Sci., 27, a viral protein required for viral DNA replication during latent infection. 2375–2380. J. Virol., 63, 2644–2649. 27. Gahn,T.A. and Sugden,B. (1995) An EBNA-1-dependent enhancer acts 17. Durocher,Y., Perret,S., Thibaudeau,E., Gaumond,M.H., Kamen,A., from a distance of 10 kilobase pairs to increase expression of the Stocco,R. and Abramovitz,M. (2000) A reporter gene assay for high- Epstein–Barr virus LMP gene. J. Virol., 69, 2633–2636. throughput screening of G-protein-coupled receptors stably or transiently 28. Mackey,D. and Sugden,B. (1999) Applications of oriP plasmids and their expressed in HEK293 EBNA cells grown in suspension culture. mode of replication. Methods Enzymol., 306, 308–328. Anal. Biochem., 284, 316–326. 29. Ambinder,R.F., Mullen,M.A., Chang,Y.N., Hayward,G.S. and 18. Chittenden,T., Frey,A. and Levine,A.J. (1991) Regulated replication of an Hayward,S.D. (1991) Functional domains of Epstein–Barr virus nuclear episomal simian virus 40 origin plasmid in COS7 cells. J. Virol., 65, antigen EBNA-1. J. Virol., 65, 1466–1478. 5944–5951. 30. Längle-Rouault,F., Patzel,V., Benavente,A., Taillez,M., Silvestre,N., 19. Yates,J.L., Warren,N. and Sugden,B. (1985) Stable replication of Bompard,A., Sczakiel,G., Jacobs,E. and Rittner,K. (1998) Up to 100-fold plasmids derived from Epstein–Barr virus in various mammalian cells. increase of apparent gene expression in the presence of Epstein–Barr virus Nature, 313, 812–815. oriP sequences and EBNA1: implications of the nuclear import of 20. Massie,B., Dionne,J., Lamarche,N., Fleurent,J. and Langelier,Y. (1995) plasmids. J. Virol., 72, 6181–6185. Improved adenovirus vector provides herpes simplex virus ribonucleotide 31. Zabner,J., Fasbender,A.J., Moninger,T., Poellinger,K.A. and Welsh,M.J. reductase R1 and R2 subunits very efficiently. Biotechnology, 13, 602–608. (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. 21. Ruponen,M., Yla-Herttuala,S. and Urtti,A. (1999) Interactions of J. Biol. Chem., 270, 18997–19007. polymeric and liposomal gene delivery systems with extracellular 32. Pollard,H., Remy,J.S., Loussouarn,G., Demolombe,S., Behr,J.P. and glycosaminoglycans: physicochemical and transfection studies. Escande,D. (1998) Polyethylenimine but not cationic lipids promotes Biochim. Biophys. Acta, 1415, 331–341. transgene delivery to the nucleus in mammalian cells. J. Biol. Chem., 273, 22. Schiffer,S.G., Foley,S., Kaffashan,A., Hronowski,X., Zichittella,A.E., 7507–7511. Yeo,C.Y., Miatkowski,K., Adkins,H.B., Damon,B., Whitman,M. et al. 33. Godbey,W.T., Wu,K.K. and Mikos,A.G. (1999) Tracking the intracellular (2001) Fucosylation of Cripto is required for its ability to facilitate nodal path of poly(ethylenimine)/DNA complexes for gene delivery. Proc. Natl signaling. J. Biol. Chem., 276, 37769–37778. Acad. Sci. USA, 96, 5177–5181. 23. Heinzel,S.S., Krysan,P.J., Calos,M.P. and DuBridge,R.B. (1988) Use of 34. Boussif,O., Zanta,M.A. and Behr,J.P. (1996) Optimized galenics improve simian virus 40 replication to amplify Epstein–Barr virus shuttle vectors in vitro gene transfer with cationic molecules up to 1000-fold. Gene Ther., in human cells. J. Virol., 62, 3738–3746. 3, 1074–1080. 24. Wysokenski,D.A. and Yates,J.L. (1989) Multiple EBNA1-binding sites 35. Boshart,M., Weber,F., Jahn,G., Dorsch-Hasler,K., Fleckenstein,B. and are required to form an EBNA1-dependent enhancer and to activate a Schaffner,W. (1985) A very strong enhancer is located upstream of an minimal replicative origin within oriP of Epstein–Barr virus. J. Virol., 63, immediate early gene of human cytomegalovirus. Cell, 41, 521–530. 2657–2666. 36. Brightwell,G., Poirier,V., Cole,E., Ivins,S. and Brown,K.W. (1997) 25. Reisman,D. and Sugden,B. (1986) trans-Activation of an Epstein–Barr Serum-dependent and cell cycle-dependent expression from a viral transcriptional enhancer by the Epstein–Barr viral nuclear antigen 1. cytomegalovirus-based mammalian expression vector. Gene, 194, 115–123. Mol. Cell. Biol., 6, 3838–3846. 37. Belting,M. and Petersson,P. (1999) Intracellular accumulation of secreted 26. Sugden,B. and Warren,N. (1989) A promoter of Epstein–Barr virus that proteoglycans inhibits cationic lipid-mediated gene transfer. Co-transfer can function during latent infection can be transactivated by EBNA-1, of glycosaminoglycans to the nucleus. J. Biol. Chem., 274, 19375–19382.

Journal

Nucleic Acids ResearchOxford University Press

Published: Jan 15, 2002

There are no references for this article.