FreeStyle™ Protein Expression Systems

Rapid, scalable, high-yield protein production from transiently transfected suspension cultures.

  • An optimized expression system for CHO and 293 suspension cultures
  • Functional proteins with mammalian posttranslational modifications
  • Easy scale-up and purification with serum-free and animal origin–free components
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The FreeStyle™ MAX System for CHO or 293 cells

The FreeStyle™ MAX System is a breakthrough protein production technology for easily scalable, rapid generation of posttranslationally modified, functional proteins in Chinese Hamster Ovary (CHO) or human embryonic kidney (HEK) 293 cells.. The cells are adapted to suspension growth in FreeStyle™ CHO or FreeStyle™ 293 Expression media, which provide an optimal balance in supporting high cell growth and maximal transfection efficiency. Transient transfection of either cell line is accomplished using the new high-efficiency FreeStyle™ MAX Reagent.

Fig 1
The choice of either CHO or 293 cells allows greater flexibility to use the most appropriate cell line to produce your recombinant protein of interest. The FreeStyle™ MAX System for CHO cells reliably produces milligram quantities of human IgG and other proteins (Table 1), and provides an advanced alternative to transient 293 expression systems and conventional stable cell line generation (Figure 1 click thumbnail image to view ).


The FreeStyle™ MAX Expression System kits include FreeStyle™ MAX Reagent, GIBCO® FreeStyle™ Expression Media, FreeStyle™ CHO or 293 cells , and OptiPRO™ SFM. You can also purchase the system components separately. The media and transfection reagent components of the FreeStyle™ MAX System are animal origin free and protein free.

Table 1 - High protein yield in FreeStyle™ MAX CHO suspension cells.

ProteinDay 1Day 2Day 3Day 4Day 5Day 6
Human IgG61828364247
Factor IX0.71.62.22.02.32.2
HGH50109178
214
225
243
EPO2.3
6.5
7.4
8.5
8.8
8.9

Human IgG, Factor IX, human growth hormone (HGH), and erythropoietin (EPO) were produced in FreeStyle™ CHO-S cells. Cells were cultured to a density of 1 x 106 cells/ml in 30 ml of FreeStyle™ CHO Expression Medium in a 125 ml shaker flask. The transfection complexes were formed using 1.25 μg of plasmid DNA and 1.25 μl of FreeStyle™ MAX Reagent per ml of culture volume to be transfected, in OptiPRO™ SFM. At 24 hours post-transfection, transfection efficiency was 60% as measured by GFP expression. Protein production was measured by ELISA, in mg/L.

The FreeStyle™ 293 System for 293 cells

The FreeStyle™ 293 System for easily scalable, rapid protein production generates functional, posttranslationally modified, proteins in human HEK293 cells. The system offers exceptional productivity and speed in the transient expression of recombinant antibody proteins (Table 2). Transfection is performed using the high-efficiency 293fectin™ transfection reagent. The transfection protocol can be readily scaled with minimal or no modifications for suspension culture volumes from less than 30 milliliters to greater than 10 liters while maintaining equivalent volumetric protein yields (Figure 3).

The FreeStyle™ 293 System kit includes 293fectin™ transfection reagent, GIBCO® FreeStyle™293 Expression Media, FreeStyle™ 293 cells , and Opti-MEM® I medium. You can also purchase the system components separately.

Table 2 - High protein yield in FreeStyle™ 293 suspension cells using 293fectin™ reagent.

ProteinDay 1Day 2Day 3Day 4Day 5Day 6
Human IgG12
63
99
128
159
164
Factor IX0.340.73
0.92
0.98
1.0
1.1
HGH19
15067
143
163
181
EPO0.03
0.05
0.08
0.1
0.11
0.14

Human IgG, Factor IX, human growth hormone (HGH), and erythropoietin (EPO) were produced in FreeStyle™ 293-F cells. Cells were cultured to a density of 1 x 106 cells/ml in 30 ml of FreeStyle™ 293 Expression Medium in a 125 ml shaker flask. The transfection complexes were formed using 1.0 μg of plasmid DNA and 2.0 μl of 293fectin™ reagent per ml of culture volume to be transfected, in OptiPRO™ SFM or Opti-MEM® I medium. At 24 hours post-transfection, transfection efficiency was >75% as measured by GFP expression. Protein production was measured by ELISA, in mg/L.

Convenience and speed

Large-scale, transient transfection of mammalian cells in suspension culture is a reliable and rapid method to generate milligram quantities of mammalian recombinant proteins for functional, therapeutic, and structural studies. With the FreeStyle™ MAX and FreeStyle™ 293 System,  you only need a sterile work bench, a CO2 incubator, and a platform shaker with shake flasks or a spinner culture. Stable cell line development requires labor-intensive screening and adaptation procedures to identify highly expressing clones.

Fig 2
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Fig 2
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The overall process requires months from the initial selection of a stably transfected cell line to production of final product. In comparison, the FreeStyle™ System achieve large-scale protein production in days, and is easily scalable from 30 ml to 10 L cultures (Figure 2 & 3 click thumbnail image to view). Detailed protocols for scale-up are available at here.

Protein production services

FreeStyle™ Custom Services are also available to produce your proteins under optimized conditions in suspension CHO or293 systems based on the scale you request. We offer timecourse studies, and protein production in 1–50 L cultures. We can provide you with the cell pellet, or even better, the purified protein, as the final deliverable. Contact your Invitrogen Sales Representative to discuss your needs and to initiate a project.
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