FC expression purification
Expression and purification of Fc fusion in 293F or 293FT:
Developed by (Mingqian Feng, 2009-05-10)
Cell maintenance:
Both 293F and 293FT can be maintained in adhesion and suspension culture, and transfered back and forth freely.
For suspension subculturing, both of them use FreeStyle 293 expression media, but 500ug/ml G-418 should be supplemented to maintain the existance of T-antigen in 293FT.
For adhesion subculture, both of them use D-MEM, but 500 ug/ml G418 is needed for 293FT.
Both 293F and 293FT are recommended to be cultured at 8% CO2.
Suspension Subculture method:
1. For T-175 vented flask, put 30 ml prewarmed FreeStyle 293 expression media.
2. Collect the seed cells by gently patting the flask, such that ~80-90 of the cells are single.
3.Spin down, resuspend the seed cells at a final density of 3x10^6/ml.
4. Add 3ml of seed cells to the T-175 flask, place it to the incubator at 8% CO2.
5. 48h later, the cell density should be 0.8-1.2x10^/ml (generally 1.0x10^6/ml).
6. Split the cells exactly every 2 days no matter what the cell density at that time.
Adhension Subculture method:
Same as Suspension Subculture Method, except for using D-MEM media instead of FreeStyle 293 Expression Media.
Suspension Transfection Method:
1. Collect the cells.
For one standard transfection, 30 million cells are needed. Resuspend the cell into 28ml of prewarmed FreeStyle 293 expression media in a 125ml SpinFlask (250ml, 500ml CellSpinFlask may be needed for large scale transfection). Put the cells at 37C while preparing DNA/293Fectin complex (next step).
2. Prepare DNA/293Fectin complex.
Dilute 30 ug plasmid into 1 ml of prewarmed OptiMem;
Dilute 60 ul 293Fectin into 1 ml of prewarmed OptiMem, immediately mix them, incubate at hood exactly 5 min.
Mix DNA solution and 293Fectin solution, incubate at hood for 25min.
3. Transfection
25 min later since the DNA/Liposome complex formation, gradually drop the complex into cell suspension while whirling the cell suspension.
4. Culture and harvest
Put the transfected cell at spin platform at a rotation speed of 75rpm, 8% CO2. The expression can be detected next day by sandwitch ELISA (generally 100-600 ng/ml at D1)
It's optional but recommended that collect the supenatant and refresh the media At D3, by doing this more protein will be achieved.
If the no changing of the media, the peak of Fc fusion concentration generally appears at D6 (seldomly at D5), thus harvested at D6.
5. Harvest
Collect the supernatant by spin down at 1000-3000rpm, filtered with 0.22um sterile membrane, put at 4C until purification (at least stable for several weeks, no sodium azide needed after filtration).
Adhension Transfection Method:
At the splitting day or the next day, add the DNA/293Fectin, for one T-175 flask, add 30 ug of DNA/60 ul of 293 Fectin.
Purification of Fc fusion protein.
1. Equilibrate the protein A column (GE, HiTrap Protein A HP, Cat. 17-0402-01) with 10 volumes of PBS 1
2. Load the supernatant by a pump (GE, Pump P-50), 1 ml/min.
3 .Wash the column with 10 volumes of PBS, 1ml/min,
4. Elute the column with Buffer solution pH2.0 ïŒSigma, Cat.82564-1L), 1ml/min, collect 1ml/tube containing 300 ul of 1 M tris-HCl (pH8.0). The target protein should appear at the 8th elution volume