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

Abstract

A scalable transfection procedure using polyethylenimine (PEI) is described for the human embryonic kidney 293 cell line grown in suspension. Green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) were used as reporter genes to monitor transfection efficiency and productivity. Up to 75% of GFP-positive cells were obtained using linear or branched 25 kDa PEI. The 293 cell line and two genetic variants, either expressing the SV40 large T-antigen (293T) or the Epstein-Barr virus (EBV) EBNA1 protein (293E), were tested for protein expression. The highest expression level was obtained with 293E cells using the EBV oriP-containing plasmid pCEP4. We designed the pTT vector, an oriP-based vector having an improved cytomegalovirus expression cassette. Using this vector, 10- and 3-fold increases in SEAP expression was obtained in 293E cells compared with pcDNA3.1 and pCEP4 vectors, respectively. The presence of serum had a positive effect on gene transfer and expression. Transfection of suspension-growing cells was more efficient with linear PEI and was not affected by the presence of medium conditioned for 24 h. Using the pTT vector, >20 mg/l of purified His-tagged SEAP was recovered from a 3.5 l bioreactor. Intracellular proteins were also produced at levels as high as 50 mg/l, representing up to 20% of total cell proteins.

Figure 1

Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear (A) or branched (B) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml^–1) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

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 µg of DNA and 2 µg of linear PEI and SEAP activity measured 72 hpt. The pEGFP plasmid (0.1 µg) 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.

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 µg of DNA and 2 µg of linear PEI and SEAP activity measured 72 hpt. The pEGFP plasmid (0.1 µg) 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.

Figure 3

Effect of serum on transgene expression. 293E cells were transfected with pTT/sgGFP (A) or pTT/SEAP (B) vectors using 1.0 µg of DNA and 2.0 µg of linear PEI (hatched boxes) or 1.5 and 2.0 µg of branched PEI (gray 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 later to a final concentration of 1%. GFP-positive cells and SEAP activity were measured 72 hpt.

Figure 4

Transfection of suspension growing cells. Cells were resuspended in 10 ml of fresh HSFM containing 1% BCS to a density of 1 × 10⁶ ml^–1 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 µg of linear or branched PEI was added to 1 ml of HEPES-supplemented HSFM containing 18 µg of pTT/SEAP and 2 µg of pEGFP or 27 µg of pTT/SEAP and 3 µg of pEGFP, respectively. Open symbols, linear PEI; closed symbols: branched PEI.

Figure 5

Effect of cell density and of conditioned medium. (A) Transfection efficiency and relative total GFP expression (in percent) obtained following transfection using standard conditions (hatched bars: 10 ml of cells at 1 × 10⁶ ml^–1 followed by addition of 10 ml of fresh medium 3 h after transfection) or using cells at 5 × 10⁵ ml^–1 in 20 ml of culture medium (gray bars). GFP was monitored 72 hpt. Relative total GFP was obtained following multiplication of percent GFP-positive cells by the mean fluorescence intensity. (B) Cells were seeded in 20 ml of 1% BCS-supplemented HSFM at a density of 2.5 × 10⁵ ml^–1 24 h before transfection. The medium was then left unchanged (conditioned: open circles) or replaced with 20 ml of fresh medium (closed circles). Three hours later, cells were transfected by the addition of 2 ml of DNA–PEI complexes (20 µg of pTT/SEAP and 40 µg of linear PEI).

Figure 6

Transient transfection in a 3.5-l bioreactor. (A) 293E cells were seeded at a density of 2.5 × 10⁵ ml^–1 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 µg pEGFP plasmids) was added to the bioreactor (unbroken lines). One hour later, 25 ml of culture was withdrawn from the bioreactor and transferred in a shake flask as a control (dashed lines). SEAP activity (circles) and GFP-positive cells (squares) were determined as described in Materials and Methods. (B) Growth curves (diamonds), viability (triangles) and yO₂ (gray line) in the 3.5-l bioreactor (unbroken lines) and shaker flask (dashed lines).

Figure 7

SEAP purification and production of other secreted and intracellular r-proteins. (A) SEAP purification by IMAC. One liter of culture medium from the 3.5-l bioreactor harvest (Fig. 6) was loaded onto a TALON™ IMAC column (10 ml bed volume). Following extensive washing, bound material was eluted with 150 mM imidazole (20 ml). Ten microliters of culture medium (lane 1), flow-through (lane 2) and eluted material (lane 3) were resolved in 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 half was transferred onto a nitrocellulose membrane and probed with anti-Myc antibody (right panel). (B) Expression of secreted C-terminal Myc-(His)₆-tagged r-proteins in a 14-l bioreactor. Lane 1, human Neuropilin-1 (1–824; upper 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). Transfections were performed as described in Materials and Methods and culture medium harvested 120 hpt. Fifteen microliters of culture medium was loaded per lane and tagged proteins detected using anti-Myc antibody. (C) Expression of intracellular r-proteins. Lane 1, pTT/sgGFP; lane 2, pTT/RR1; lane 3, pTT empty vector; lane 4, pcDNA3.1/G_αq; lane 5, pTT/G_αq; lane 6, pTT/p27^Kip1; lane 7, pTT/PYC; lane 8, pTT/E1B^19K; lane 9, pTT/hexokinase; lane 10, pTT/glucokinase. Cells were harvested 72 hpt, rinsed with PBS and solubilized in NuPAGE sample buffer followed by sonication (lanes 1–5) or extracted in lysis buffer (lanes 6–10) as indicated in Materials and Methods. Proteins were resolved on a 4–12% Bis–Tris NuPAGE gradient gel and stained with Coomassie blue R-250.