Editing FC expression purification

Expression and purification of Fc fusion in 293F or 293FT: 

Developed by (Mingqian Feng, 2009-05-10)

'''Cell maintenance:'''<br> 

Both 293F and 293FT can be maintained in adhesion and suspension culture, and transfered back and forth freely. <br> 

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.

<br> For adhesion subculture, both of them use D-MEM, but 500 ug/ml G418 is needed for 293FT. <br> 

Both 293F and 293FT are recommended to be cultured at 8% CO2.



'''Suspension Subculture method:'''<br> 1. For T-175 vented flask, put 30 ml prewarmed FreeStyle 293 expression media.<br> 2. Collect the seed cells by gently patting the flask, such that ~80-90 of the cells are single. <br> 3.Spin down, resuspend the seed cells at a final density of 3x10^6/ml.<br> 4. Add 3ml of seed cells to the T-175 flask, place it to the incubator at 8% CO2.<br> 5. 48h later, the cell density should be 0.8-1.2x10^/ml (generally 1.0x10^6/ml). <br> 6. Split the cells exactly every 2 days no matter what the cell density at that time.

'''Adhension Subculture method:'''<br> Same as Suspension Subculture Method, except for using D-MEM media instead of FreeStyle 293 Expression Media.

'''Suspension Transfection Method:'''<br> 1. Collect the cells. <br> 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&nbsp; be needed for large scale transfection). Put the cells at 37C while preparing DNA/293Fectin complex (next step).<br> 

2. Prepare DNA/293Fectin complex. <br> Dilute 30 ug plasmid into 1 ml of prewarmed OptiMem; <br> Dilute 60 ul 293Fectin into 1 ml of prewarmed OptiMem, immediately mix them, incubate at hood exactly 5 min.<br> Mix DNA solution and 293Fectin solution, incubate at hood for 25min.<br>

3. Transfection<br> 25 min later since the DNA/Liposome complex formation, gradually drop the complex into cell suspension while whirling the cell suspension.<br>

4. Culture and harvest<br> 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)

<br> It's optional but recommended that collect the supenatant and refresh the media At D3, by doing this more protein will be achieved. <br> If the no changing of the media, the peak of Fc fusion concentration generally appears at D6 (seldomly at D5), thus harvested at D6. 

<br> 5. Harvest <br> 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).

<br>'''Adhension Transfection Method:'''

<br> 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.'''

<br> 1. Equilibrate the protein A column (GE, HiTrap Protein A HP, Cat. 17-0402-01) with 10 volumes of PBS 1<br> 2. Load the supernatant by a pump (GE, Pump P-50), 1 ml/min.<br> 3 .Wash the column with 10 volumes of PBS, 1ml/min,<br> 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<br><br>