Fecturin™ is ideal for the production of recombinant protein in HEK-293 and derivatives as well as in CHO cells grown either adherently or in suspension in synthetic media. This reagent is extremely gentle to cells, a strong advantage upon adaptation to suspension culture. This in turn increases protein yields dramatically. In addition, transfection efficiency with Fecturin™ ranges between 75 – 85 % (Fig. 1).

Figure 1. Transfection of HEK-293 and CHO using Fecturin™. Cells were seeded in either FS or C-CHO media, two commercially available synthetic media. HEK-293 (left) or CHO (right) cells were transfected with pCMV-EGFP plasmid following the standard Fecturin™ protocol. Cells were visualized by fluorescent microscopy 24 h after transfection.

In order to match stringent regulatory requirements in production laboratories, Fecturin™ is chemically defined and guaranteed free of any component of animal origin. Additional QC certificates may be provided upon request. Fecturin™ of various quality grades, including qualified for drug biomanufacturing, is also available upon request.

High levels of secreted VEGF (400 ng VEGF/106 cells/day) are obtained following transfection of HEK-293 with Fecturin™ in synthetic media. Moreover, Fecturin™ compares favourably to the commercially available reagent LP1 (Fig. 2).

Figure 2. Compaison of protein expression levels after transfection of HEK293 cells with Fecturin™ and LP1 reagent. Suspension cells were seeded in FS medium and transfected with pCMV- Luc plasmid using Fecturin™ or LP1 following the manufacturer’s recommendations. Luciferase protein content is quantified 24 h post-transfection.

Fecturin™mediated transfection results in high levels of protein production in CHO suspension culture when compared to LP2 lipid-based reagent, in two different commercially available synthetic media dedicated to CHO cells (Fig. 3). Up to 9 µg of luciferase/106 cells/ml/day is produced using Fecturin™ in C-CHO medium.

Figure 3. Compaison of protein expression levels after transfection of suspension CHO cells with Fecturin™ and LP2 in two commercially available synthetic media. Cells were seeded in either I-CHO or C-CHO synthetic media and transfected with pCMV-Luc plasmid using either Fecturin™ or LP2 following the manufacturer’s recommendations. Luciferase expression (RLU) was assayed 24h after transfection.

Upon transfection of CHO cells with Fecturin™, the daily expression of secreted VEGF (Vascular Endothelial Growth Factor) increases over four days (Fig. 4).

Figure 4. Daily VEGF production in suspension CHO cells. Cells were seeded in C-CHO synthetic medium and transfected with pCMV-VEGF plasmid using Fecturin™ VEGF protein production was quantified by ELISA.

The cumulative VEGF production also increases steadily over 9 days (Fig. 5).

Figure 5. Cumulative VEGF production in CHO cells in suspension. Cells were seeded in C-CHO synthetic medium and transfected with pCMV-VEGF plasmid using Fecturin™. VEGF protein production was quantified by ELISA.

Protein production in suspension culture of CHO cells can be easily scaled-up with Fecturin™. This reagent has been used to produce VEGF in 500 ml suspension culture of CHO cells with yields up to 10 µg of VEGF/106 cells /ml /day (Fig. 6). Protein production yield per ml increases significantly as the cell culture volume is scaled up. Protocols for 1 L or more are available upon request from our technical support.

Figure 6. VEGF production in increasing volumes of culture. CHO cells were seeded in increasing volumes of C-CHO synthetic medium and transfected with pCMV-VECG plasmid using Fecturin™. Amount of VEGF protein produced was quantified by ELISA, 2 days after transfection.

Increase the yield of recombinat proteins with Fecturin transfection reagent in serum free, suspension culture.
Polyplus News # 2 p. 6-9, 2006. (636 KB)