293F Protein Production

Expression and purification of Fc fusion in 293F or 293FT: (Mingqian Feng, 2009-05-10)

Cell maintenance and Culture

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.

1. Remove the cryovial of cells from the liquid nitrogen and thaw quickly in a 37°C water bath.

2. Just before the cells are completely thawed, decontaminate the outside of the vial with 70% ethanol. Gently break up clumps and cell pellet if present and transfer the entire contents of the cryovial into a 125 ml polycarbonate, disposable, sterile Erlenmeyer shaker flask containing 30 ml of pre-warmed FreeStyle 293 Expression Medium.

3. Incubate cells in a 37°C incubator containing a humidified atmosphere of 8% CO2 in air on an orbital shaker platform rotating at 135 rpm.

4. Next day, determine viable and total cell counts. Generally, viability is > 70%; a bit lower is no reason for concern, but if viability is less than 60% thaw a new batch of cells.

5. Subculture the FreeStyle 293-F cells when they reach a density between 1 x 10^6 and 3 x 10^6 viable cells/ml, usually 24 -48 hours, seed shaker flasks at 0.3 x 10^6 viable cells/ml in pre-warmed FreeStyle 293 Expression Medium. Use 125 or 250 ml polycarbonate, disposable, sterile, Erlenmeyer flasks containing 40 or 80 ml total working volume of cell suspension, respectively.

Freeze FreeStyle 293-F cell line at a density of 1 x 10^7 viable cells/ml with a freezing medium composed of 90% fresh FreeStyle 293 Expression Medium and 10% DMSO. The freezing rate should be a decrease of 1°C per minute. Transfer frozen vials to liquid nitrogen for long-term storage.

Suspension Culture

Use polycarbonate, disposable, sterile Erlenmeyer flask with vented cap for suspension culture
For culture volumes above 400 ml, lower the speed of the orbital shaker if foam  is generated. Use 90 rpm for 1 L culture.

At high stirring speeds (i.e. greater than 130 rpm) and/or depending on the impeller design, Add Pluronic® F-68 (2.5-5 ml/L of 10% Pluronic® F-68, Invitrogen Catalog no. 24040) to FreeStyleTM 293 Expression Medium.

Do not add antibiotics to media as this may negatively impact cell growth.

Flasks

Flask Volume	Culture Volume	Manufacturer	Catalog no
125 ml	25 - 40 ml	VWR	30180-036
250 ml	50 - 80 ml	VWR	30180-044
500 ml	100 – 200 m	VWR	30180-052
1 liter	200 – 400 ml	VWR	82013-164
3 liter	600 – 1000 ml	Corning	431252

Spinner Flask

the optimum spinner speed was 100-130 rpm and 70-100 rpm impeller speed in Celligen stirred tank bioreactors. Seed cells at 0.3-0.5 x 10^6 viable cells/ml.

Suspension Subculture method (Mingqian Feng):

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 3 x 10^6/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 0.8-1.2x10^6/ml (generally 1.0x10^6/ml).
6. Split the cells exactly every 2 days no matter what the cell density at that time.

Adhesion Culture

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.

Adhension Subculture method (Mingqian Feng):
Same as Suspension Subculture Method, except for using D-MEM media + 10% FBS  instead of FreeStyle 293 Expression Media.

Use Adhesion Culture Cells for Suspension Transfection

1. Grow cells in DMEM + 10% FBS for fast growth. Split cells every 2 or 3 days.

2. The day before transfection, remove medium (remove as much as possible), rinse cells with DPBS, remove DPBS.

3. Digest cells with Trypin (as little as possible). Add 5 to 10 ml FreeStyle medium, pipet up and down to break cells aggregate into single cells.

4. Transfer to 250 ml culture flask. Add FreeStyle medium to dilute to 6~7 x 10^5 cells/ml.

5. The cell density should be around 1X 10^6 cells/ml. Add more medium if cell density is > 10^6 cells/ml.

6. Transfect cells using PEI MAX or GeneExpressoMax.

Suspension Transfection Method

1. Collect the cells.

Approximately 24 hrs before transfection, pass FreeStyle 293-F cells at 6~7 x 10^5 cells/ml. Place the flask(s) on an orbital shaker platform rotating at 135 rpm at 37°C, 8% CO2. Do not add antibiotics during transfection.

On the day of transfection, the cell density should be about 1.2-1.5 x 10^6/ml.

For one standard transfection, 30 million (3 x 10^7) cells are needed (from one T75 flask). Resuspend the cell in 30 ml of prewarmed FreeStyle 293 expression media in a 125ml SpinFlask (250ml, 500ml Cell SpinFlask may  be needed for large scale transfection).

For 100 ml culture, 100 million (10X10^7) cells from one T175 flask plus one T75 flask are required. Use 1 liter VWR flask for culture.

Put the cells at 37 ^oC while preparing DNA/GeneExpressoMax complex (next step).

2. Prepare DNA/293Fectin GeneExpressoMax complex.
Dilute 30 ug plasmid into 1 ml of prewarmed OptiMem DMEM serum-free medium; 

Dilute 60 ul 293Fectin GeneExpressoMax into 1 ml of prewarmed OptiMem DMEM serum-free medium, immediately mix them, incubate at hood exactly 5 min, 2 min.

To transfect cells from one T175 flask+ one T75 flask: dilute 100 ug DNA in 3 ml serum-free DMEM. Dilute 300 ul GeneExpresso in 3 ml serum-free DMEM.

Mix DNA solution and 293Fectin GeneExpresso solution, incubate inside the hood for 25 20 min.

3. Transfection

After incubate for 20 min, 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 75 rpm, 8% CO₂. The expression can be detected next day by sandwitch ELISA (generally 100-600 ng/ml at D1).

Yield per ml at D1:

30 ml: 3 to 18 ug/30 ml

Yield per ml at D3:

5~20 ug/ml for most proteins

100 ug/ml for short peptide Fc fusion.

1 ug/ml or less for difficult protein.

Expected Total Yield at D3:

30 ml: 150 ug to  600 ug

100 ml: 500 ug to 2 mg

Very difficult protein: ~ 100 ug or less from 100 ml.

For low expression protein: Culture the cells up to 7 days. There is no need to change or supplement the culture medium during the first 6 to 7 days.

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 harvest at D6.

5. Harvest
Collect the supernatant by spin down at 1000-3000rpm, filtered with 0.22um sterile membrane, Store at 4 ^oC (cold room) until purification (at least stable for several weeks, no sodium azide needed after filtration).

	Cells from Adhesion Culture Flasks	DNA	Transfection Reagent	Dilution in DMEM	0.5 M VPA (ul)	Freestyle Medium (ml)
30 ml Transfection	Three  T75 flasks	30 ug	90 ul of 1 mg/ml	2 X 1 ml	225	30
100 ml Transfection	One T175 + One T75	125 ug	375 ul of 1 mg/ml	2 X 3 ml	750	100

Further Optimization needed:

DNA amount, cell density, and volume of Freestyle medium needed for sustain growth and protein production

Adhension Transfection Method

At the splitting day or the next day, add the DNA/293Fectin, GeneExpresso for one T-175 flask, add 30 ug (can be vary from 24-42 ug) of DNA/60 ul of  293 Fectin. GeneExpressonMax

Transfect with PEI MAX

PEI: (Linear: Polysciences cat # 23966 or PEI Max: 24765-2). Dissolve in 25mM HEPES and 150 mM NaCl (pH 7.5), 1 mg/ml. Sterile filtered using a 0.22 μM syringe filter, aliquoted, and frozen at -20°C.

Valproic acid (VPA) (Sigma cat. No. P4543-100G). 250 mM VPA sodium salt in water, filter through 0.22 μM. Store at -20 oC.

DNA to PEI ratio: 1:3

Amount:

DNA: 3 μg/ml
PEI: 9 μg/ml

1.  24 hours prior to transfection, split cells to a density of ~1 x 10^6 cells/ml, in 50 ml medium, and culture overnight in the CO2 incubator with shaking at 37°C. Cell density should be ~2 x 10^6 at the time of transfection.

2. Count cells using a hemocytometer.

3. Spin down (1000 rpm, 2 min) a sufficient volume to resuspend the cells at a density of 2.5 x 10^6 cells/ml in fresh 293F Freestyle Media in the desired volume for transfection.

Transfections should be performed at a cell density of between 2.5 x 10^6 and 3.0 x 10^6 cells/ml.

For example:

culture density of the suspension culture stock = 2.8 x 10^6 cells/ml.
For a final transfection volume of 100 ml,  50 ml of cells at a density of 2.5 x 10^6 cells/ml is needed.
V1 = 2.8 x 10^6 cells/ml = 50 ml X 2.5 x10^6 cells/ml
V1 = 44.6 ml
Spin 44.6 ml at 1000 rpm for 2 min and remove media from cell pellet.
Resuspend cells in 50 ml of fresh pre-warmed Freestyle 293 medium.
Return the culture to the CO2 incubator while preparing DNA and PEI mixtures for transfection. Reserve an aliquot of the culture for cell counting to confirm cell density.

4. For 50 ml transfection:
Add 150 μg DNA to ~ 250 μl of pre-warmed DMEM medium (final DNA conc is 0.5 μg/μl)

Dilute 1mg/ml PEI  to 0.5 μg/μl in DMEM. Final amount is 450 μg PEI. So add 450 μl  1mg/ml PEI to 450 μl DMEM.

5. Mix DNA with PEI solution, incubate for 20 min.

6. Add to cells. Return cells to incubator, .

7. After 24 hr, dilute the cells 1:1 with (i.e., 50 ml) pre-warmed Freestyle 293 Medium supplemented with final conc of 2.5 mM VPA (add 500 μl of 250 mM VP stock solution to 50 ml pre-warmed Freestyle 293 Medium, and add this medium to culture). (For 293E cells, optimal conc is 3.75 to 4 mM of VPA).

8. Harvest the transfected cells 3-6 days after transfection. Protein production can be monitored by taking small samples (~100 μl) at 24 hr intervals, clarifying the media by centrifugation and analysis by SDS-PAGE  to determine the optimal time for harvest.

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 (Sigma, Cat.82564-1L), or adjust 0.2 M Glycine with 6 N HCl to pH 2.0, 1ml/min, collect 1ml/tube containing  300 ul of 1 M Tris-HCl (pH8.0), or 80 ul of 1 M Tris (pH not adjusted).The target protein should appear at the 8th elution volume

Large Scale Production

Materials

Cell Culture

1. HEK-293 cells adapted to cultivation in serum-free suspension
2. Cylindrical and square-shaped glass bottles with nominal volumes of 100 mL to 5 L
3. Ex-cell 293 medium without L-glutamine and phenol red (Sigma-Aldrich).
4. 50× L-glutamine and phenol red solution. A stock solution with 200 mM glutamine and 250 g/mL phenol red is made by dissolving 29.23 g/L glutamine  and 250 mg phenol red (Sigma-Aldrich) in 800 mL water. After the compounds are dissolved, the volume is adjusted to 1 L by further addition of water. The solution is sterilized by filtration through a 0.2- um Steritop (bottle-top) filter unit  with a neck size of 45 mm (Millipore). The solution is transferred into sterile 50-mL centrifuge tubes and kept frozen at −20°C. For each liter of Ex-cell® 293 medium, 20 mL of the stock solution is added.
5. Trypan blue solution (0.4%) (Sigma-Aldrich).

Transfection

1. RPMI 1640 medium containing 25 mM HEPES and 4 mM glutamine (Lonza).
2. Pluronic® F-68 dissolved in water at a concentration of 2%. The solution is filter sterilized and stored at room temperature.
3. VPA (2-propyl-pentanoic acid, sodium salt) (Sigma-Aldrich) is prepared as a 500-mM solution in water, filter sterilized, aliquoted into sterile 50-mL tubes, and stored at −20°C. The working solution is maintained at 4°C. VPA is a known teratogen.

4. Linear 25 kDa polyethyleneimine (PEI) (Polysciences) is dissolved in water at 1 mg/mL at a pH of 7.0. When dissolving, lower the pH with 1 N HCl. When the PEI is in solution, the pH is increased to 7.0 with  1 N NaOH. The solution is filter sterilized, aliquoted into sterile 50-mL tubes, and stored at −20°C. It can be stored frozen for years as long as repeated freeze–thaw cycles are avoided.
5. Ex-cell® 293 medium without L-glutamine and phenol red
6. 50× L-glutamine–phenol red solution.

IgG Purification

1. Streamline-rProtein A beads (GE Healthcare).
2. Econo-Pac chromatography column (Bio-Rad).
3. Equilibration and washing buffer: 20 mM sodium phosphate (pH 7.5).
4. Elution and regeneration buffer: 100 mM sodium citrate (pH 3).
5. Neutralizing buffer: 1 M Tris–HCl (pH 8.0).
6. Storage buffer: 20% ethanol in 20 mM sodium phosphate (pH 7.5).
7. Centricon Plus-70 (Millipore).
8. Phosphate buffered saline (PBS) at pH 7.1.

ELISA

1. 96-well ELISA microtiter plates with flat bottom.
2. Blocking buffer: 0.5% casein hydrolysate (Applichem) and 0.05% Tween 20 (Sigma-Aldrich) in PBS (pH 7.1).
3. Capture antibody: Goat anti-human kappa light chain (AbD Serotec).
4. Coating solution: For each 96-well plate, 11 L of capture antibody is mixed with 11 mL of PBS (pH 8.0).
5. Washing buffer: PBS (pH 8.0) with 2% Tween 20.
6. Detection antibody: Alkaline phosphatase-conjugated goat anti-human gamma heavy chain (Biosource).
7. Standard: Human IgG, whole molecule (ChromPure, Jackson ImmunoResearch). The standard is diluted in blocking buffer to 40 ng/mL and then serially
diluted 1:2 with blocking buffer.
8. Substrate buffer: Add 97 mL diethanolamine (Sigma-Aldrich) to 700 mL H2O and adjust pH to 9.8 with 2 M HCl. Add 0.5 mL 1 M MgCl2 and 2 g NaN3. Adjust volume to 1 L.
9. Substrate: 4-nitrophenyl phosphate disodium salt (NPP) (Applichem) is dissolved in substrate buffer to 1.5 mg/mL.
10. Stop solution: 3 M NaOH.

Methods

Cell Culture

1. HEK-293 cells are subcultivated every 3–4 days by inoculation in 100 mL Ex-cell® 293 medium (when used for cell culture, the medium contains L-glutamine and phenol red in a 250-mL square-shaped glass bottle at an initial cell density of 0.3 × 10^6 cells/mL.
2. Determine the cell density and viability by trypan blue staining using a Neubauer hemocytometer chamber and an inverted phase contrast microscope.
3. After cell counting, transfer 3–4 × 107 cells into a 50-mL centrifuge tube and centrifuge at 500 × g for 5 min in a standard tabletop centrifuge.

4. The medium is removed by aspiration or decanting. The cell pellet is resuspended in 10 mL of Ex-cell® 293 medium and transferred to a 250-mL square-shaped bottle containing 90 mL of prewarmed Ex-cell® 293 medium.
5. Attach the bottle to a platform mounted on an orbital shaker using double-sided adhesive transfer tape (3M) and agitate at 110 rpm at 37°C in a 5% CO2 atmosphere without humidity. Keep the cap of the bottle opened about one quarter of a turn.

Cell Expansion for Transfection

1. Count cells one day before transfection.
2. Transfer 6 × 10^8 cells into a 250-mL centrifuge bottle. For this step, it is necessary to have two 100-mL cultures.
3. Centrifuge the cells for 10 min at 1,000 rpm at room temperature.
4. Remove the medium by aspiration or decanting and gently resuspend the cell pellet in 50 mL of prewarmed Ex-cell® 293 medium.
5. Transfer 25 mL into each of two 1-L square-shaped glass bottles with 275 mL of Ex-cell® 293 medium. The starting cell density of each culture is about 1 × 10^6 cells/mL.
6. Place the bottles on an orbital shaker and incubate at 37°C overnight with agitation at 110 rpm. Keep the bottle caps open one quarter of a turn.

Transfection

1. Count cells in the two 1-L bottles.
2. Transfer a total of 1 × 10^9 (1 billion) cells from the two overnight cultures into two 500-mL centrifuge bottles (Corning) and centrifuge at 1,000 rpm for 10 min at room temperature.
3. Remove the medium by aspiration or decanting and resuspend the cells from the two centrifuge bottles in a total volume of 50 mL by addition of prewarmed RPMI 1640 medium containing 0.1% Pluronic® F-68. The cell density after resuspension is 20 × 10^6 (20 million) cells/mL.
4. Transfer the cells to a 250-mL cylindrical glass bottle.
5. Add 1.25 mg of plasmid DNA to the culture and mix gently by swirling the bottle. For the example described here, 612 ug of pXLGHEK-RhLC, 612 ug pXLGHEK-RhHC, and 25 ug pEGFP-N1 (49:49:2 w/w/w ratio) are added to the culture.

6. Add 3.75 mL linear 25-kDa PEI solution (1 mg/mL) to the culture and gently mix by swirling the bottle.
1 billion cells:  1.25 mg DNA + 3.75 mg PEI ---> 50 ml of 20 million cells/ml. 'PEI to DNA ratio is 3'.

7. The bottle is agitated in an incubator shaker at 110 rpm at 37°C in a 5% CO2 atmosphere without humidity with the bottle cap slightly opened.
8. After 3 h of incubation, transfer the cells to a 5-L cylindrical glass bottle containing 950 mL of prewarmed Ex-cell® 293 medium.

Final volumn is 1000 ml.
9. Add 7 mL of 0.5 M VPA to achieve a final concentration of 3.75 mM.
10. Incubate the culture as in step 7.
11. After 1 day of incubation, the analysis of transfection efficiency is performed on a small aliquot (20 uL) by flow cytometry on a Guava EasyCyte (Millipore). The sample is diluted 1:10 in PBS and the percentage of GFP-expressing cells is determined by counting at least 10,000 cells.

Small Scale (10 mL transfection)

Resuspend 10 × 10^6 cells in 0.5 mL of RPMI 1640

Add 12.5 ug plasmid DNA and 37.5 uL PEI (1 mg/mL stock solution).

After transfection add 9.5-mL Ex-cell 293 medium.

Add 70 uL of 0.5 M VPA 24 hours later.

Medium Scale (100 mL transfection)

Resuspend 100 × 10^6 cells in 5 mL of RPMI 1640.

Add 125 ug plasmid DNA and 375 uL PEI (1 mg/mL stock solution).

After transfection add  95 mL Ex-cell 293 medium.

Add 700 uL of 0.5 M VPA 24 hours later.

Analysis of Antibody Production

1. To measure recombinant antibody accumulation over time, 100 uL aliquots are taken daily during the production phase. After centrifugation to remove cells, the antibody concentration in each sample is measured by sandwich ELISA.
2. Coat a 96-well ELISA plate overnight at 4°C with 100 uL of goat anti-human kappa light chain IgG diluted in PBS (coating solution).
3. Remove the coating solution with a multichannel pipettor and wash each well three times with 200 uL of washing buffer. The final wash is performed just
before the samples are loaded into the wells. After the final wash, tap the plate on a paper towel to remove any remaining wash solution.
4. Samples from the culture are diluted 1:10 in blocking buffer, and then 200 uL of each is loaded in triplicate onto the plate. Two serial two-fold dilutions in blocking buffer (100 uL sample + 100 uL blocking buffer) are done directly on the plate.
5. Load the antibody standard as serial 1:2 dilutions in triplicate on the plate.
6. Incubate the plate for 1 h at 37°C and then remove the samples using a multichannel pipetter.
7. Wash each well three times with 200- uL washing buffer as in step 3.
8. Add AP-conjugated goat anti-human gamma chain IgG diluted 1,000-fold in PBS to each well. For each plate, dilute 11 uL of antibody in 11 mL of PBS and add 100 uL to each well.
9. Incubate the plate for 1 h at 37°C.
10. Remove solutions and wash each well three times in 200 uL washing buffer as in step 3.
11. Add 100 L substrate solution to each well and cover the plate with aluminum foil.
12. Incubate the plate for 15 min at room temperature with gentle agitation.
13. Stop the reaction by addition of 100 L of 3 M NaOH.
14. Measure the absorbance at 405 nm using a microplate reader.
15. Determine the antibody concentration in each sample after generation of the standard curve from the absorbance of the standard antibody samples.

Harvest and Purification of the Recombinant Antibody

1. At the end of the production phase of 7–12 days, cells are harvested by transferring the culture into two 500-mL centrifuge bottles (Corning).
2. Centrifuge at 2,000 × g for 15 min at 4°C.
3. Recover the supernatant by decanting into a 1-L bottle.

4. Remove any additional cell debris by filtration with a 500-mL filter unit with a 0.45- m membrane (Corning) and transfer the filtrate into two 500-mL centrifuge bottles (Corning).

If protein has 6His tag, Ni beads can be used for purification: dialyse filtered medium against 25 mM phosphate buffer saline (PBS, pH8.0) at 4 °C o/ before loading to Ni column.

5. Equilibrate the Streamline-rProtein A beads. For 1 L of supernatant containing about 1 g of antibody, pipette 20 mL beads into a 250-mL centrifuge bottle. Wash the beads three times by addition of 200 mL of 20 mM sodium phosphate (pH 7.5) to the bottle. Mix gently by inverting several times. Let the
beads settle to the bottom of the bottle and carefully remove most of the buffer by decanting, paying attention not to lose the beads.
6. With a 10-mL pipette, transfer the washed beads along with the remaining buffer to the two 500-mL centrifuge bottles with the cell culture medium and incubate overnight with agitation on an STR4 rotator at 4°C.
7. Let the beads settle by gravity to the bottom of the conical centrifuge bottles and then transfer them to a chromatography column with a 10-mL pipette. Let the beads deposit at the bottom of the column. Remove the tip of the column to allow liquid flow.
8. After the medium has drained from the column, wash the beads with 10 column volumes (CVs) of 20 mM sodium phosphate (pH 7.5) and collect the flow-through in a clean beaker.
9. Elute the antibody from the beads with 5 CVs of 100 mM sodium citrate (pH 3.0) and collect in a 250-mL bottle.
10. Add one volume of 1 M Tris–HCl (pH 8.0) to the eluate. Store at 4°C until the solution is ready to be concentrated.
11. Regenerate the column with 5–10 CVs of 100 mM sodium citrate buffer (pH 3.0).
12. Reequilibrate the column with 5 CVs of 20 mM sodium phosphate (pH 7.5).
13. Remove the beads from the column by adding 20 mM sodium phosphate buffer (pH 7.5) with 20% ethanol and pouring them in a 50-mL centrifuge tube.
14. Store at 4°C.
15. Concentrate the partially purified antibody and change the buffer to PBS using a Centricon Plus-70 (Millipore) according to the manufacturer’s protocol.
16. Determine the antibody concentration by measuring absorbance at 280 nm. Convert absorbance units to concentration with the conversion factor (1 U A280 = 1 mg/mL antibody).

Transfect 293T or 293F cells using PEI (not suspension culture)

1. Extract plasmid with chloroform to kill bacteria.

2. Add 1 mg DNA to 100 ml serum free DMEM.

3. Add 2 ml 1 mg/ml PEI 25K.

4. Incubate 10 min at RT.

5. Add to one HYPERFlask containing  450 ml  DMEM + 2% FBS.--> Final is 1.6% FBS

Triple flasks: 150 μg DNA + 250 μl PEI in 30 ml serum free DMEM-->70 ml DMEM + 2% FBS

HYPERFlasks: 1 mg DNA + 2 ml PEI. (in 100 ml serum free DMEM)-->450 ml DMEM + 2% FBS

2125 cm2 expanded surface roller bottle (Greiner): 0.5 mg DNA + 50 ml serum-free DMEM, mixed, + 1 ml PEI stock (1 mg/ml), add to roller bottle containing 200 ml of DMEM, 2% FBS.

6. Collect conditioned medium   4–7 days after transfection.

7. Filter through a 500-mL filter unit with a 0.45- m membrane (Corning) and transfer the filtrate into two 500-mL centrifuge bottles (Corning).

If protein has 6His tag, Ni beads can be used for purification: dialyse filtered medium against 25 mM phosphate buffer saline (PBS, pH8.0) at 4 °C o/ before loading to Ni column.

8. Purify protein using Ni beads or protein A beads.