High-yield production of human Dicer by transfection of human HEK293-EBNA1 cells grown in suspension

Bouvette, Jonathan; Korkut, Dursun Nizam; Fouillen, Aurélien; Amellah, Soumiya; Nanci, Antonio; Durocher, Yves; Omichinski, James G.; Legault, Pascale

To achieve high-level expression of recombinant human Dicer, we relied on transient transfection of a suspensiongrown HEK-293 cell line expressing the Epstein-Barr virus antigen-1 (HEK293-EBNA1 or 293-6E) with a pTT5 expression vector carrying a CMV promoter and the Epstein-Barr virus origin of replication oriP [31] [32] [33] . This mammalian expression system has been engineered for maximal expression of recombinant His-tagged proteins in serum-free media [34] . Linear polyethyleninime (PEI) was selected as an efficient and cost-effective transfection reagent. For optimization of transfection conditions, small-scale suspension cultures were grown in serum-free media, either as 2-mL cultures in 6-well plates or 20-mL cultures in 20-mL disposable Erlenmeyer flasks. In initial experiments where different quantities of plasmid DNA (1, 1.5 and 2 mg per L of culture) and PEI:DNA ratios (2:1 or 3:1) were tested, we found that optimal transfection efficiency could be reached when transfecting cells diluted to 0.8 × 10 6 cells/mL 24 h in advance using 1 mg of Dicer-expressing pTT5 plasmid with 2 mg linear PEI per L of culture. To estimate the transfection efficiency, we replaced 5% of the total transfected plasmid by a GFP-expressing plasmid and used fluorescence microscopy to count the percentage of GFP-expressing cells. Transfection efficiencies of 30-40% were typically reached at 48 h post transfection (hpt) (Fig. 1a) . Such transfection efficiencies are considered very good especially when taking into account that they are underestimated compared to a transfection using 100% GFP-expressing plasmid [35] . The cell cultures were monitored up to 96 hpt to assess cell viability and viable cell density as well as the intracellular expression of recombinant Dicer. It was found that cell viability decreases below 75% beyond 72 hpt (Fig. 1b, green) , whereas viable cell density is maintained at 3.2 × 10 6 cell/mL (Fig. 1b, black) and Dicer expression levels are not significantly changed (Fig. 1c) . Similar results were obtained with large-scale transfections of up to 1 L. Thus, the optimum time for collecting the cells for protein purification was determined to be 72 hpt, taking into consideration that additional time may lead to protein modifications that would adversely affect its activity. At 72 hpt, volumetric expression of the intracellular Dicer protein was 14 ± 4 mg per L of culture on average (Fig. 1c) .