Editing 293E Protein Production

== 293E Cell culture<br>  ==

All reagents added to cultures and media must be sterile. 

'''Calf serum, cosmic '''(HyClone) 

Thaw the frozen serum thoroughly at 37°C. Heat-inactivate for 30 min in a 56°C bath, swirling the bottle occasionally for thorough heat distribution. Store aliquots at -20°C. 

'''Cell lines''', 293E or 293-6E (NRC-BRI) 

Other HEK293 cell lines (e.g., 293T, 293S, and 293F) can also be used, but with somewhat lower r-protein yields. 

Use appropriate culture medium listed below, depending on cells used. 

Culture medium, prewarmed, for 293E cells (e.g., '''LC-SFM medium''' [custom low-calcium HSFM formulation; Invitrogen]) 

Supplement with '''1% (v/v) serum and 0.1% (w/v) Pluronic F-68 '''prior to use. 

<br> 

Culture medium, prewarmed, for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone]) 

<br> 

The best media for 293E expression is:<br> 

1:1 mixture of Free Style and SFM4Transfx-293 plus 100 ug/ml G418<br> 

'''Free Style'''<br> 

Cat #: 12338026, 12338-026, 6X1 L, $519<br> 

http://www.lifetechnologies.com/order/catalog/product/12338018<br> 

SFM4Transfx-293<br>Cat #: SH30860.LS, 6 x 1L<br>http://www.thermoscientific.com/en/product/hyclone-sfm4transfx-293-media.html<br><br><br> 

Add '''10''' mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use. 

DMSO, cell culture grade (Sigma) 

G418, 50 mg/mL stock solution (Invitrogen) 

caution PBS containing 25 mg/mL '''Erythrosin B''' (Sigma) 

'''Pluronic F-68''', '''10%''' stock solution (w/v) (Invitrogen)

<br>

== Optimized Condition for Gag-VLP<br> ==

<br>[[Image:Culture_medium2.jpg]]<br> 

[[Image:Culture_medium.jpg]]<br>

<br>

[[Image:Insulin_transferrin_lipidmix.jpg]]<br>

<br>

<br>

<br>

<br>

1. Transfection<br>

Grow cells in mid-log phase (2~3 X10^ cells/ml), spin down, use new medium and do transfection with 25 kDa L-PEI (1 ug DNA + 2 ug PEI), Dilute DNA and PEI in culture medium. 1 ug DNA per ml cells. Incubate DNA-PEI for 15 min then add to cells.<br>

Best cell density for transfection: 10^6 cells/ml for freestyle medium (mid-log phase 2.9 X 10^6 cells/ml), and 2X10^6 for supplemented freestyle (5.4 X10^6 cells/ml)

2. Harvest: <br>

2 or 3 days after transfection, spin down at 500 g for 15 min at 4 oC, collect supernatant.<br>

3. Spin down at 25000 rpm (115,254 xg) for 3 hours at 4 oC using a SW32 rotor.<br>

5. Resuspend in cool PBS (250 ul per tube), incubate at 4 oC for 2 hour.<br>

6. Add Benzonase, 200 U/ml,&nbsp; treat for 1 hour at room temperature.<br>

7. Concentrate VLP by spin ultrafiltration column. Load to 130 ml Sepharose 6FF (XK 16/70 column ), using PBS. Flow rate is 2 ml/min. (60 cm/h), 2.5 ml/tube.&nbsp;<br>

8. Filter through 0.2 uM . Add Trehalose to 15%. Filter with 0.2 uM filter and store at 4 oC.<br>

Should be ~7000 copies of Cre/FLP per Gag VLP<br>



Supplements:

Lipids: 100X, 100X Synthecol, Sigma. Fatty acids, 100X F7050, Sigma. Tocopherol acetate, 0.2 mg/ml, T1157 Sigma. Emulsifying agent polysorbate 80 (2.5 mg/ml, P4780, Sigma



=== Equipment  ===

Rinse glassware three times using Milli-Q H2O (or equivalent), followed by steam sterilization. 

Boxes, Styrofoam (large and 20-position small) or programmable cooler (see Steps 12 and 16) 

Centrifuge 

Cryovials 

Flasks, Erlenmeyer, glass, reusable (Corning) 

Flasks, Erlenmeyer, plastic, 125-mL disposable shake (Corning) 

Freezer at -80°C 

Freezer storage, liquid nitrogen, vapor phase 

Hemocytometer 

Hood, laminar flow 

Incubator preset to 37°C, humidified, 5% CO2 

Microscope 

Shaker, orbital 

Ensure that the orbital shaker can operate continuously under humidified conditions. Do not shut off the orbital shaker in the humidified incubator or it will seize upon subsequent use. When not in use for cultures, leave the shaker agitating or remove it from the incubator until needed. Avoid shakers with digital controls, as they are susceptible to humidity. The shaker must contain holders for shake flask sizes ranging from 50 to 2000 mL. If no holders are supplied, equip the platform with an anti-slip adhesive mat. Shake flasks must stay on the platform up to '''120 rpm'''. 

Tubes, conical, 15-mL 

Water bath preset to 37°C<br>Previous SectionNext Section<br><br> 

=== METHOD  ===

Conduct all operations aseptically in a laminar flow hood. Cells growing in serum-free media are highly sensitive to chemical contamination; thorough rinsing of all equipment that contacts the cells with highly pure water is compulsory.<br> 

'''Cell Thawing and Maintenance''' 

1. Prepare a 15-mL conical tube containing 10 mL of prewarmed cell culture medium appropriate for the cell line to be used. 

2. Thaw the cells rapidly in a 37°C water bath. 

3. Add the cells to the 15-mL tube. Invert to mix. 

4. Dilute a 100-μL aliquot of cells with 100 μL of PBS containing 25 mg/mL '''erythrosin B'''. Determine the cell density and viability using a hemocytometer. 

5. Centrifuge the cells at '''200g''' for 5 min at room temperature. 

6. Decant the supernatant. Loosen the pellet by gently tapping the tube. Dilute the cells to 2.5 × 10<sup>5</sup> cells/mL in a 125-mL disposable shake flask using cell culture medium. 

Operating culture volumes must be in the range of 16%-33% of the nominal shake flask volume for sufficient oxygenation. 

7. Place the shake flask on the orbital shaker (~120 rpm) in the incubator. 

Center the shaker in the incubator. Make sure that the walls, door, and wire are not contacted during agitation. 

8. Count the cells at 48 h post-thawing. If necessary, dilute to 2.5 × 105 cells/mL using cell culture medium. 

9. When the doubling time is 24 h for two consecutive passages, subculture every 2 or 3 d to maintain cell densities between 2.5 × 105 and 1.2 × 106 cells/mL using medium supplemented with G418 (25 μg/mL or 50 μg/mL, for 293-6E or 293E cells, respectively). 

See Troubleshooting. 

10. Dilute the cultures to 1.5 × 10<sup>5</sup> or 7.5 × 10<sup>4 </sup>cells/mL for weekends or long weekends (3 or 4 d, respectively). 

'''Cell Freezing''' 

11. Prepare the freezing mixture by adding DMSO to fresh cell culture medium (10:90, v/v). 

12. Label the cryovials. Place in a small Styrofoam box. Prechill the large Styrofoam box in a -80°C freezer. 

13. Count the cells. Determine the volume needed for cryopreservation. 

Cells must be in the exponential phase (between 8 × 10<sup>5</sup> and 1.2 × 10<sup>6</sup> cells/mL). 

14. Centrifuge the cells at 200g for 5 min. Decant the supernatant. Dissociate the pellet by gently tapping the tube. 

15. Add the freezing medium drop-wise to the cells while swirling the tube to obtain the desired density (i.e., 5 × 10<sup>6</sup> to 5 × 10<sup>7</sup> cells/mL per vial). 

16. Quickly aliquot the cells into the labeled vials. Immediately transfer to a -80°C freezer. Do not open the freezer door for at least 2 h. 

Alternatively, freeze cells at 1°C/min in a programmable cooler. 

17. Transfer the vials to liquid nitrogen freezer storage (vapor phase) the following day. 

Store for a maximum of 4 d. 

'''''<br>TROUBLESHOOTING''''' 

Problem: The cells do not grow well. 

[Step 9] 

Solution: If cells do not grow with the expected doubling time, consider the following: 

1. The cultureware is not sufficiently clean. Glassware for all culture manipulations and reagent preparations must be rinsed at least three times with high-purity H2O. 

2. The H2O used may not be sufficiently pure. Check the source of the ultrapure H2O. 

3. Check that the filtration system (if applicable) used to make the ultrapure H2O is functioning properly. 

4. Cell aggregation can give a false impression of slower growth rates. Check regularly that the orbital shaker is at the right speed and that the incubator is at the correct temperature and is supplying the prescribed amount of CO2. 

5. The cells may be too old. The number of cell passages indicates the age of the cells in service. A new cryovial can be thawed every 3 mo, on average. 

6. Verify expiration dates, culture medium lots, and batches of additives used (e.g., Pluronic F-68 and antibiotics). 

7. Check for mycoplasma contamination; if positive, thaw new cells. 

<br> 

=== Culture  ===

=== <br>  ===

1. One day before transfection pass HEK-293E cells&nbsp; in Ex-cell 293 serum-free medium with the final cell density of 1x106 cells/ml. <br> 

2. Before transfection determine the cell number in the suspension culture using PCV <br> 

3. Spin down 20x10^6 cells and resuspend in 0.89 ml of RPMI 1640 (Invitrogen, Carlsbad, CA).<br> 

4. Transfect cells with DNA (1.5 μg/1x10^6 cells) + PEI (3 to 4.5 μg/10^6 cells). Incubate cells with DNA-PEI for 3 hours.<br> 

5. Dilute cells with fresh Ex-Cell 293 serum-free media (Sigma, cat. no. 14571C) to the final cell density of 1x10^6 cells/ml.<br> 

6. Add VPA to final conc of 3.75 mM 24 hours post transfection. 

7. Havest cells at 3 to 7 days. 

ESF serum free medium (Expression Systems cat. no. 98-001) can also be used? 

== Transfection of HEK293-EBNA1 Cells in Suspension with 293fectin for Production of Recombinant Proteins  ==

=== <br>'''INTRODUCTION'''<br>  ===

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin, due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a “must-have” technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, other methods suitable for efficient transfection of mammalian cells are easier to use. This protocol describes the steps needed for successful transfection of 293-6E cells in suspension culture in serum-free medium using 293fectin.<br><br> 

=== <br>'''MATERIALS'''  ===

'''Reagents'''<br> 

*293fectin (Invitrogen)<br> 
*293-6E HEK293-EBNA1 cells, maintenance (in shake flask). The presence of G418 does not interfere with transfection.<br> 
*Culture medium for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone])<br>

Add 10 mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use.<br> 

*Opti-MEM I medium (Invitrogen)<br> 
*Plasmid DNA of interest, purified<br> 
*Plasmid, fluorescent protein-expressing <br>

Adding a plasmid encoding a fluorescent protein (e.g., green fluorescent protein [GFP]) to 5% in the transfection mixture does not significantly alter expression of the gene of interest and allows visual (or quantitative, if using flow cytometry) confirmation of transfection efficiency. A few GFP-positive cells can be detected as early as 3-4 h post-transfection using a fluorescence microscope.<br> 

*Pluronic F-68, 10% stock solution (w/v) (Invitrogen)TN1 medium, prewarmed (e.g., FreeStyle medium or LC-SFM containing 20% [w/v] Tryptone N1 [Organotechnie]) (optional; see Step 12)Supplement with 0.1% (w/v) Pluronic F-68 and filter-sterilize prior to use.

<br> '''Equipment'''<br> 

Containers, plastic (e.g., Tupperware)<br> 

Flasks, Erlenmeyer, shake, plastic disposable 125-mL (Corning)<br> 

Hemocytometer<br> 

Incubator preset to 37°C, humidified, 5% CO2Microscope, fluorescence <br> 

Paper towels<br> 

Plates, six-well, disposable (Nunc)<br> 

Shaker, orbital<br> 

Tubes, 1.5- or 15-mL<br> 

Vortex mixer<br><br>'''METHOD'''<br> 

'''Small-Scale Transfection in Six-Well Plates or 125-mL Shake Flasks using 293fectin'''<br> 

Although the protocol described here uses a DNA:293fectin ratio of 1.5:1.3, the ratio selected for production should be determined by testing various ratios in six-well plate experiments. These small-scale tests must include regular samplings to determine the optimal harvest periods. The flask procedure is scaleable to 650 mL in a 2-L shake flask. <br> 

1. One or two days prior to transfection, dilute the cells to'''2.5 × 105 cells/mL (for 48 h prior) or 5 × 10<sup>5</sup> cells/mL (for 24 h prior)''' with cell culture medium. Optimal cell density should be reached on the day of transfection. '''This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection.''' <br> 

<br> 

2. On the day of transfection, warm the Opti-MEM I medium to 25°C-37°C. Remove the 293fectin from the refrigerator. Remove the DNA from the freezer to thaw. <br> 

<br> 

3. From a maintenance flask, determine the cell density and viability using a hemocytometer. Cell density at transfection should range from 8 × 10<sup>5</sup> to 1.2 × 10<sup>6</sup> cells/mL, provided the doubling time is 24 h and viability is greater than 95%. <br> 

<br> 

4. Add 1.8 mL of cells to each of the six wells (or 18 mL to a new 125-mL shake flask). Transfer to an incubator. <br> 

To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. <br> 

<br> 

5. Add 100 μL of Opti-MEM I to each of six 1.5-mL tubes (or 1.0 mL to a 15-mL tube for a 125-mL shake flask). <br> 

<br> 

6. Add the required DNA volume corresponding to 3 μg to each tube (or 30 μg to the 15-mL tube). Vortex. <br> 

To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. <br> 

<br> 

7. To another 1.5-mL tube, add 682 μL of Opti-MEM I and 18 μL of 293fectin (or to another 15-mL tube, add 1.0 mL Opti-MEM I and 26 μL 293fectin). Vortex gently. <br> 

<br> 

8. Incubate for 5 min at room temperature. <br> 

<br> 

9. Add 100 μL of diluted 293fectin to each DNA-containing tube (or the entire 1.0 mL of the 293fectin dilution to the 15-mL DNA tube). Vortex immediately after addition. <br> 

<br> 

10. Incubate the mixtures for 20 min at room temperature. <br> 

<br> 

11. Remove the culture (from Step 4) from the incubator. Add the DNA/293fectin mixture(s) and swirl. Return the culture to the incubator. <br> 

<br> 

12. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells (or 500 μL for the 125-mL shaker flask). Return the culture to the incubator. The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production. <br> 

<br> 

13. Harvest the r-proteins: <br> 

For intracellular r-proteins <br> 

*i. Harvest the cells at 48-72 h post-transfection. Establish the best harvesting time by performing an expression kinetics study. For secreted r-proteins <br> 
*ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing. Since culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. <br>

<br> '''TROUBLESHOOTING'''<br> 

''Problem'': The transfection does not work.[Step 13]<br> 

''Solution'': Consider the following: <br> 

*1. Make sure the cells are in exponential growth phase for transfections. If the culture grows to higher-than-prescribed transfection densities, medium nutrient depletion and metabolic by-products will compromise the transfection. <br> 
*2. Anti-clumping agents such as dextran sulfate can inhibit transfection. <br> 
*3. DNA plasmid quality may be inadequate. Check for contaminants by A260/A280 ratio readings; acceptable values range between 1.80 and 1.95. In addition, the DNA plasmid can be digested and run on an agarose gel to verify its integrity (it should be mostly in a supercoiled form) and/or to detect RNA contamination. 
*4. Monitor cell line productivity occasionally by transfecting the cells with an easily measured protein such as secreted alkaline phosphatase or GFP. Low transfection efficiencies are readily indicated when the percentage of GFP-positive cells is below 30% by 48 h post-transfection. Also, for each new medium or reagent lot, monitor transfection efficiencies using six-well plate assays.

== <br>'''Small-Scale Transfection in Six-Well Plates or 125-mL Shake Flasks using Branched PEI'''  ==

INTRODUCTION 

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a “must-have” technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. In particular, the branched isoform of PEI works well for adherent cells, as it promotes their attachment to the plastic surface. It is thus very useful in experiments requiring multiple medium exchanges or washing steps following transfection. Also, when used in conjunction with six-well CellBIND plates, branched PEI can be used to adhere transfected cells when establishing stable cell lines. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in adherent culture using branched PEI.<br><br> 

=== <br>MATERIALS<br>  ===

<br>'''Reagents''' 

293E cells, maintenance (in T-flask or shake flask) 

See Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins (this issue) for preparation. The presence of G418 does not interfere with transfection. 

Calf serum, cosmic (HyClone) 

Thaw the frozen serum thoroughly at 37°C. Heat-inactivate for 30 min in a 56°C bath, swirling the bottle occasionally for thorough heat distribution. Store aliquots at -20°C. 

G418, 50 mg/mL stock solution (Invitrogen) 

LC-SFM medium (Invitrogen) 

Supplement with 1% (v/v) heat-inactivated calf serum and 50 μg/mL G418 prior to use. 

cautionPhosphate-buffered saline (PBS) 

cautionPolyethyleneimine (PEI), branched (1 mg/mL) 

Plasmid DNA of interest, purified 

Prepare using CSH Protocols articles Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation or Preparation of Plasmid DNA by Large-Scale Boiling Lysis. 

Plasmid, fluorescent protein-expressing (optional; see Step 8) 

Adding a plasmid encoding a fluorescent protein (e.g., green fluorescent protein [GFP]) to 5% in the transfection mixture does not significantly alter expression of the gene of interest and allows visual (or quantitative, if using flow cytometry) confirmation of transfection efficiency. A few GFP-positive cells can be detected as early as 3-4 h post-transfection using a fluorescence microscope. 

Pluronic F-68, 10% stock solution (w/v) (Invitrogen) 

TN1 medium, prewarmed (e.g., FreeStyle medium or LC-SFM containing 20% [w/v] Tryptone N1 [Organotechnie]) (optional; see Step 13) 

Supplement with 0.1% (w/v) Pluronic F-68 and filter-sterilize prior to use.<br>'''Equipment''' 

Containers, plastic (e.g., Tupperware) 

Hemocytometer 

Incubator preset to 37°C, humidified, 5% CO2 

Microscope, fluorescence (optional; see Step 8) 

Paper towels 

Plates, six-well, disposable (CellBIND; Corning) 

Tubes, 15-mL 

Vortex mixer<br> 

=== <br><br>METHOD  ===

<br> 

Although the protocol described here uses a DNA:PEI ratio of 1.5:2, the ratio selected for production should be determined by testing various ratios in six-well plate experiments. These small-scale tests must include regular samplings to determine the optimal harvest periods. 

1. Warm the LC-SFM culture medium to 37°C. 

2. Remove the cell maintenance T-flask from the incubator. Dislodge the cells by gently tapping the side of the flask 

This is not necessary if the maintenance cultures are in shake flasks. 

3. Remove a cell aliquot to determine cell density and viability using a hemocytometer. 

4. In a 15-mL tube, dilute the cells to 2.5 × 105 cells/mL in a volume of 12 mL of medium. 

This can be scaled down to 1 mL in 12-well plates. 

5. Add 1.8 mL of cells to each of the six wells. Transfer to the incubator. Culture for 24 h. 

To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. 

6. On the day of transfection, warm the PBS to 25°C-37°C. Thaw the DNA and PEI. 

7. Aliquot 100 μL of PBS to each of six 1.5-mL tubes. 

8. Add 3 μg of DNA to each tube. Vortex. 

To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. 

9. To another 1.5-mL tube, add 672 μL of PBS. Add 28 μL of branched PEI solution. Vortex. 

10. Add 100 μL of PEI solution to each DNA-containing tube. Vortex immediately (three times, 3 sec each) after PEI addition. 

Compare different DNA:PEI ratios to see whether different lots and/or production batches affect transfection efficiency and productivity. Each new PEI preparation must be tested. 

11. Incubate for 15 min at room temperature. 

12. Remove the six-well plate from the incubator. Add the DNA/PEI mixture to each well and swirl the plate. Return the plate to the incubator. 

13. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells. Return the culture to the incubator. 

The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production. 

14. Harvest the r-proteins: 

For intracellular r-proteins 

i. Harvest the cells at 48-72 h post-transfection. 

Establish the best harvesting time by performing an expression kinetics study. 

For secreted r-proteins 

ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing. 

Because culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. 

See Troubleshooting. 

<br>TROUBLESHOOTING 

Problem: The transfection does not work. 

[Step 14] 

Solution: Consider the following: 

1. Make sure the cells are in exponential growth phase for transfections. If the culture grows to higher-than-prescribed transfection densities, medium nutrient depletion and metabolic by-products will compromise the transfection. 

2. Anti-clumping agents such as dextran sulfate can inhibit transfection. 

3. DNA plasmid quality may be inadequate. Check for contaminants by A260/A280 ratio readings; acceptable values range between 1.80 and 1.95. In addition, the DNA plasmid can be digested and run on an agarose gel to verify its integrity (it should be mostly in a supercoiled form) and/or to detect RNA contamination. 

4. Monitor cell line productivity occasionally by transfecting the cells with an easily measured protein such as secreted alkaline phosphatase or GFP. Low transfection efficiencies are readily indicated when the percentage of GFP-positive cells is below 30% by 48 h post-transfection. Also, for each new medium or reagent lot, monitor transfection efficiencies using six-well plate assays. 

<br> 

== Transfection of HEK293-EBNA1 Cells in Suspension with Linear PEI for Production of Recombinant Proteins  ==

=== <br>'''INTRODUCTION'''  ===

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Pure r-proteins are often required in large amounts (hundreds of milligrams to gram quantities) when being developed as biotherapeutics, or in smaller quantities (milligrams) for high-throughput screening campaigns and structural or functional studies. Mammalian cells are often preferred over prokaryotic systems when expressing cDNAs of mammalian origin due to their superior capability to conduct elaborate post-translational modifications. Large-scale transfection of mammalian cells is now establishing itself as a “must-have” technology in the scientific community, as it allows the production of milligram to gram quantities of r-proteins within a few days after cDNA cloning into the appropriate expression vector. Although calcium-mediated large-scale transfection is very effective, it is usually achieved in serum-containing medium under tightly controlled conditions that are difficult to achieve on a large scale. In contrast, polyethylenimine (PEI) is much easier to use: It binds to and precipitates DNA efficiently and the resulting DNA-PEI complexes are suitable for efficient transfection of mammalian cells. PEI has been used successfully on a large scale in serum-containing and serum-free cultures. In particular, the linear isoform of PEI is more effective for transfecting cells in suspension. This protocol describes the steps needed for successful transfection of HEK293 cells adapted to serum-supplemented or serum-free medium in suspension culture using linear PEI.<br> 

=== <br>MATERIALS  ===

=== <br>'''Reagents'''  ===

Cell lines, 293E or 293-6E (NRC-BRI), maintenance cells 

See Culture of HEK293-EBNA1 Cells for Production of Recombinant Proteins (this issue) for preparation. The presence of G418 does not interfere with transfection. 

Use appropriate culture medium listed below, depending on cells used. 

Culture medium for 293E cells (e.g., LC-SFM medium [Invitrogen]) 

Supplement with 1% (v/v) heat-inactivated serum and 0.1% (w/v) Pluronic F-68 prior to use. 

Culture medium for 293-6E cells (e.g., F17 culture medium [Invitrogen] or HyQ SFM4Transfx-293 [HyClone]) 

Add 10 mL of Pluronic F-68 stock solution per liter of culture medium to a final concentration of 0.1% (w/v) prior to use. 

cautionPhosphate-buffered saline (PBS) 

Plasmid DNA of interest, purified 

Prepare using CSH Protocols articles Preparation of Plasmid DNA by Alkaline Lysis with SDS: Maxipreparation or Preparation of Plasmid DNA by Large-Scale Boiling Lysis. 

Plasmid, fluorescent protein-expressing (optional; see Step 6) 

Adding a plasmid encoding a fluorescent protein (e.g., green fluorescent protein [GFP]) to 5% in the transfection mixture does not significantly alter expression of the gene of interest and allows visual (or quantitative, if using flow cytometry) confirmation of transfection efficiency. A few GFP-positive cells can be detected as early as 3-4 h post-transfection using a fluorescence microscope. 

Pluronic F-68, 10% stock solution (w/v) (Invitrogen) 

cautionPolyethylenimine (PEI), linear (1 mg/mL) 

TN1 medium, prewarmed (e.g., FreeStyle medium or LC-SFM containing 20% [w/v] Tryptone N1 [Organotechnie]) (optional; see Step 10) 

Supplement with 0.1% (w/v) Pluronic F-68 and filter-sterilize prior to use.<br>Equipment 

Containers, plastic (e.g., Tupperware) 

Flasks, Erlenmeyer, shake, plastic, disposable (Corning) 

Hemocytometer 

Incubator preset to 37°C, humidified, 5% CO2 

Microscope, fluorescence (optional; see Step 6) 

Paper towels 

Plates, six-well, disposable (Nunc) 

Shaker, orbital 

Tubes, 1.5- or 15-mL 

Vortex mixer<br> 

=== <br>METHOD  ===

<br>Small-Scale Transfection in Six-Well Plates or 125-mL Shake Flasks 

While the protocol described here uses a DNA:PEI ratio of 1:2, the ratio selected for production should be determined by testing various ratios in six-well plate experiments. These small-scale tests must include regular samplings to determine the optimal harvest periods. The flask procedure is scalable to 650 mL in a 2-L shake flask. 

1. One or two days prior to transfection, dilute the cells to 2.5 × 105 cells/mL (for 48 h prior) or 5 × 105 cells/mL (for 24 h prior) with the appropriate cell culture medium. 

Optimal cell density should be reached on the day of transfection. This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. 

2. On the day of transfection, warm the PBS to 25°-37°C. Thaw the DNA and PEI. 

3. From a maintenance flask, determine the cell density and viability using a hemocytometer. 

Cell density at transfection should range from 8 × 105 to 1.2 × 106 cells/mL, provided the doubling time is 24 h and viability is greater than 95%. 

4. Add 1.8 mL of cells to each of the six wells (or 18 mL to a new 125-mL shake flask). Transfer to an incubator. 

To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. 

5. Add 200 μL of PBS to six 1.5-mL tubes (or 2.0 mL to a 15-mL tube for the 125-mL shake flask). 

6. Add 2 μg of DNA to each tube (or 20 μg to the 15-mL tube for the 125-mL shake flask). Vortex gently. 

To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. 

7. Add 4 μL of PEI to each tube (or 40 μL to the 15-mL tube for the shake flask) containing DNA solution. Vortex immediately (three times, 3 sec each) after PEI addition. 

Compare different DNA:PEI ratios (1:2 vs. 1:3, etc.) to see whether different lots and/or production batches affect transfection efficiency and productivity. Each new PEI preparation must be tested. 

8. Incubate the mixture for 15 min at room temperature. 

9. Remove the culture (from Step 4) from the incubator. Add the DNA/PEI mixture(s) and swirl. Return the culture to the incubator. 

10. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h post-transfection to each of the six wells (or 500 μL for the 125-mL shaker flask). Return the culture to the incubator. 

The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production. 

11. Harvest the r-proteins: 

For intracellular r-proteins 

i. Harvest the cells at 48-72 h post-transfection. 

Establish the best harvesting time by performing an expression kinetics study. 

For secreted r-proteins 

ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing. 

Since culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. 

See Troubleshooting. 

=== <br>TROUBLESHOOTING  ===

Problem: The transfection does not work. 

[Step 11] 

Solution: Consider the following: 

1. Make sure the cells are in exponential growth phase for transfections. If the culture grows to higher-than-prescribed transfection densities, medium nutrient depletion and metabolic by-products will compromise the transfection. 

2. Anti-clumping agents such as dextran sulfate can inhibit transfection. 

3. DNA plasmid quality may be inadequate. Check for contaminants by A260/A280 ratio readings; acceptable values range between 1.80 and 1.95. In addition, the DNA plasmid can be digested and run on an agarose gel to verify its integrity (it should be mostly in a supercoiled form) and/or to detect RNA contamination. 

4. Monitor cell line productivity occasionally by transfecting the cells with an easily measured protein such as secreted alkaline phosphatase or GFP. Low transfection efficiencies are readily indicated when the percentage of GFP-positive cells is below 30% by 48 h post-transfection. Also, for each new medium or reagent lot, monitor transfection efficiencies using six-well plate assays. 

<br> 

== Another protocol<br>  ==

<br>Use a DNA:PEI ratio of 1:2 to 1:3. The flask procedure is scalable to 650 mL in a 2-L shake flask. 

<br>1. One or two days prior to transfection, dilute the cells to 2.5 × 10^5 cells/mL (for 48 h prior) or 5 × 10^5 cells/mL (for 24 h prior) with the appropriate cell culture medium. Optimal cell density should be reached on the day of transfection. This avoids having to centrifuge the cells,&nbsp; which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. 

<br>2. On the day of transfection, warm the PBS to 25°-37°C. Thaw the DNA and PEI. 

<br>3. From a maintenance flask, determine the cell density and viability using a hemocytometer. Cell density at transfection should range from 8 × 10^5 to 1.2 × 10^6 cells/mL, provided the doubling time is 24 h and viability is greater than 95%. 

<br>4. Add 1.8 mL of cells to each of the six wells (or 18 mL to a new 125-mL shake flask). Transfer to an incubator.<br>To minimize medium evaporation, put the plates in a plastic container lined with a wet paper towel to preserve humidity. “Trap” some CO2 in the container before closing the lid. 

<br>5. Add 200 μL of PBS to six 1.5-mL tubes (or 2.0 mL to a 15-mL tube for the 125-mL shake flask). 

<br>6. Add 2 μg of DNA to each tube (or 20 μg to the 15-mL tube for the 125-mL shake flask). Vortex gently.<br>To evaluate transfection efficiency, add a GFP expression plasmid at 5% of the final DNA content used. A reasonable indicator of successful transfection is when the percentage of GFP-positive cells is between 30% and 50% by 48-72 h post-transfection. 

<br>7. Add 4 μL of PEI to each tube (or 40 μL to the 15-mL tube for the shake flask) containing DNA solution. Vortex immediately (three times, 3 sec each) after PEI addition. 

<br>Compare different DNA:PEI ratios (1:2 vs. 1:3, etc.) to see whether different lots and/or production batches affect transfection efficiency and productivity. Each new PEI preparation must be tested. 

<br>8. Incubate the mixture for 15 min at room temperature. 

<br>9. Remove the culture (from Step 4) from the incubator. Add the DNA/PEI mixture(s) and swirl. Return the culture to the incubator. 

<br>10. For secreted protein production, add 50 μL of prewarmed TN1 medium at 24-48 h posttransfection to each of the six wells (or 500 μL for the 125-mL shaker flask). Return the culture to the incubator. 

<br>The final TN1 concentration in the culture(s) should be 0.5% (w/v). TN1 supplementation is not necessary for intracellular protein production.<br> 

11. Harvest the r-proteins:<br>For intracellular r-proteins<br> 

i. Harvest the cells at 48-72 h post-transfection.<br>Establish the best harvesting time by performing an expression kinetics study. 

<br>For secreted r-proteins<br>ii. Harvest the supernatants between 96 and 168 h post-transfection as long as the culture viability is high and the product titer is increasing.<br>Since culture viability decreases with time, it is important to harvest when the viability is relatively high (i.e., greater than 75%) to protect the r-protein from proteolytic degradation. 

== <br><br>Purification of His-Tagged Proteins Using Fractogel-Cobalt  ==

=== INTRODUCTION  ===

Fast and efficient production of recombinant proteins (r-proteins) remains a major challenge for the academic and biopharmaceutical communities. Such proteins often need to be as pure as possible before any characterization study can begin. Although many types of protein tag are available, histidine is the most popular. Although small-scale immobilized metal-affinity column (IMAC) purification of such proteins (e.g., &lt;500 mL of culture medium) can easily be achieved using gravity chromatography columns, larger volumes can be processed with the aid of automated chromatography systems. This protocol describes an IMAC purification technique for secreted proteins using a cobalt-loaded resin. Preliminary small-scale trials using this technique can be used to determine the production scale that will be needed to provide enough pure material for a given study.<br><br> 

=== MATERIALS  ===

==== <br>Reagents  ====

All solutions (including the culture supernatant) must be filtered (0.45 μm) and degassed to minimize air bubble formation in the column. 

cautionCleaning buffer 

200 mM Cobalt chloride (CoCl2) 

cautionCobalt stripping buffer 

Culture supernatant 

For large volumes of culture medium, concentrate the clarified culture supernatant 10- to 20-fold by tangential flow ultrafiltration prior to r-protein capture on the IMAC column. 

H2O, Milli-Q-filtered 

cautionIMAC column elution buffer 

cautionIMAC wash buffer 1 

cautionIMAC wash buffer 2 

0.5 M NaCl 

0.5 M NaCl (pH 5.0 ± 0.2) 

cautionPhosphate-buffered saline (PBS) 

Protein assay reagent, 5X concentrate (Bio-Rad) 

Dilute to 1X in H2O prior to use. 

Resin, His-Bind Fractogel (M) (EMD Biosciences) 

cautionStorage buffer for IMAC 

==== <br>Equipment  ====

Column, chromatography (e.g., Qiagen-tip 500 Maxi kit [2-cm diameter, 5-mL minimum bed volume] or HiSpeed Maxi Tip [3.5-cm diameter, 10-mL minimum bed volume]) 

For small-scale trials (20-500 mL), pack a gravity column with 1-5 mL of resin, with a bed height of at least 1 cm. 

Column, desalting, disposable, prepacked (e.g., Econo-Pac 10DG; Bio-Rad) 

Microplate reader (optional; see Step 13) 

Pipette 

Plates, 96-well 

Pump, peristaltic (optional, for large volumes; see Step 6.ii) 

Spectrophotometer 

Tubes, 1.5-mL<br>Previous SectionNext Section 

=== <br>METHOD  ===

==== <br>Column Charging  ====

1. Pack a gravity column with Fractogel resin. 

Refer to the manufacturer’s binding capacity specifications in order to use an adequate volume of resin. 

2. Add five column-volumes of 0.5 M NaCl. 

3. Add four column-volumes of 200 mM CoCl2. 

4. Add two column-volumes 0.5 M NaCl (pH 5 ± 0.2). 

====  ====

==== Sample Loading and r-Protein Elution  ====

Use SDS-PAGE analysis on reserved 500-μL aliquots of the original sample feed, flowthrough, washes, and pooled protein fractions to determine whether the purification process is sufficient or whether the column resin is overloaded beyond its protein-binding capacity. 

5. Add 10 column-volumes of PBS to equilibrate the column. 

6. Load the feed onto the column: 

For small volumes (&lt; 100 mL) 

i. Allow the sample to pass through the column by gravity. 

For large volumes (&gt; 100 mL) 

ii. Attach a peristaltic pump to the column outlet to pull the sample feed through the column more quickly. 

iii. Use a flow rate up to a maximum of 10 mL/min (190 cm/h) for a 5-mL Qiagen-tip 500 column or 20 mL/min (125 cm/h) for a 10-mL HiSpeed Maxi Tip column. 

iv. Stop and disconnect the pump 1-2 min before the entire sample empties from its reservoir. 

v. Add the last few milliliters of sample to the reservoir with a pipette. Allow it to flow through by gravity. 

This prevents the column from drying out. 

7. Keep the flowthrough and store at 4°C. Retain a 500-μL aliquot for subsequent gel analysis. 

Do not discard the flowthrough or wash fractions until it has been determined that the r-protein has been entirely captured and eluted from the column. 

8. Add 10 column-volumes of IMAC wash buffer 1. Retain a 500-μL aliquot of the flowthrough for subsequent gel analysis. 

9. Add 10 column-volumes of IMAC wash buffer 2 to remove impurities bound to the column. Retain a 500-μL aliquot of the flowthrough for subsequent gel analysis. 

10. Add four column-volumes of IMAC column elution buffer in 1-mL increments. Harvest individual 1-mL fractions in prelabeled 1.5-mL tubes. 

11. Add 5-20 μL of each eluted fraction to a 96-well plate. 

12. Add 200 μL of diluted protein assay reagent to each well. 

Make sure that each sample is well mixed as the reagent is being added. 

13. Wait 5-10 min for color development. 

The wells with the darkest blue coloration correspond to the fractions containing the r-protein. For more quantitative determination, read the absorbance at 595 nm using a microplate reader. 

See Troubleshooting. 

14. Pool the 1-mL fractions containing r-protein. 

15. Desalt the purified r-protein using a desalting column equilibrated in PBS or in your preferred buffer. 

16. Based on the molar extinction coefficient of the protein, determine its concentration after measuring its absorbance at 280 nm (use an appropriate buffer as the blank). 

The molar extinction coefficient of your protein can be obtained by uploading its amino acid sequence at http://ca.expasy.org/tools/protparam.html. 

==== <br>Column Washing and Storage  ====

17. Add two column-volumes of cobalt stripping buffer to the column. 

18. Add two column-volumes of H2O. 

19. Add 15 column-volumes of cleaning buffer. 

20. Add five column-volumes of H2O. 

21. Add five column-volumes of PBS (or more if necessary) to neutralize the pH of the column to 7.0-7.2. 

22. Add five column-volumes of storage buffer for IMAC. 

23. Add an additional 1-2 mL of storage buffer for IMAC on top of the column. Cover the top and bottom. Store at 4°C. 

==== <br>TROUBLESHOOTING  ====

Problem: Purification from IMAC does not work. 

[Step 13] 

Solution: Consider the following: 

1. Perform SDS-PAGE analysis of the feed, flowthrough, washes, and pooled fractions to see whether the r-protein was captured by the resin or not. If the His-tagged protein is in the flowthrough, the tag on the protein itself may be sterically hindered and inaccessible for capture by the column. 

2. The r-protein could be aggregated, thus hampering its capture onto the column. Check this by gel filtration chromatography. 

3. Increasing the pH of the feed to 8.0 or higher can enhance r-protein binding to the column, and charging the IMAC column with nickel instead of cobalt can increase binding affinity. 

4. Sometimes, the r-protein elutes in IMAC wash buffer 2. In this case, decrease the imidazole concentration of the wash buffer. 

<br> 

<br> 

[[Category:293_Expression]] [[Category:Protocols]]