Mammalian Expression for High-Yield Protein Production Support—Getting Started

For stable high-yield expression, we offer the Freedom CHO-S and Freedom DG44 kits. For transient high-yield expression, we offer the ExpiCHO Expression System, Expi293 Expression System, FreeStyle 293 Expression System, FreeStyle Max 293 Expression System, and FreeStyle Max CHO Expression System. For high-yield expression of functional membrane proteins in Exp293F cells, we offer the Expi293 MembranePro Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293 and the technology of MembranePro to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro Functional Protein Expression System. Please see the Application Note for more details.

We recommend using the pcDNA™ 3.4-TOPO® TA vector (Cat. No. A14697). This vector contains the native, full-length CMV promoter and a WPRE (Woodchuck Posttranscriptional Regulatory Element) downstream of the cloning site, both of which contribute to high level gene expression (about 2–3 fold higher expression than with pcDNA™3.3 TOPO® vector, which in turn provides 2–5 fold higher expression than with a standard pcDNA™ vector. Of course, the expression level is also protein-dependent. 

pcDNA™ 3.4-TOPO® TA vector is an improvement over pcDNA™ 3.3-TOPO® TA vector. It contains the WPRE (Woodchuck Posttranscriptional Regulatory Element) that allows for 2- to 3-fold higher levels of expression than pcDNA™ 3.3-TOPO® TA vector.

Most 293 cell lines can be adapted directly from conventional serum-containing or other serum-free media into Expi293™ Expression Medium by either using the Direct Adaptation method or Sequential Adaptation method, described in the manual.

Note: Adapted cells may not show the same high levels of expression obtained using Expi293F™ cells.

The ExpiFectamine™ 293 Transfection Enhancers 1 & 2 are optimized cocktails of proprietary reagents designed to work with the ExpiFectamine™ 293 Reagent to increase protein expression levels and hence transient protein yields. They must be added at the appropriate time (20 hours post-transfection) to obtain maximal protein yield. Addition of enhancers earlier or later will result in lower protein yields. If you forget to add enhancers during the recommended time window, we recommend adding them as soon as possible. Omission of the enhancers from the protocol can cause protein yield to be up to 3-fold lower. The enhancers are designed to work together for maximal expression. Addition of just one enhancer will result in reduced expression and may be anywhere from one-third to two-thirds the level of expression obtained when both enhancers are added.

We do not recommend using OptiPRO™ SFM to make DNA–ExpiFectamine™ 293 transfection complexes. If you require an animal origin–free system, you may use FreeStyle™ 293 Expression Medium to make the complexes, but keep in mind that there will be a 10–20% drop in final protein yields.

The ExpiFectamine™ 293 Transfection Enhancers 1 & 2 are designed to work with the ExpiFectamine™ 293 Reagent and do not work well with other transfection reagents.

No, you cannot purchase the enhancers separately. They are only sold as part of a kit (with ExpiFectamine™ 293 Reagent).

Expi293™ Expression Medium is not directly compatible with ProBond™ or Ni-NTA purification systems. We recommend performing a buffer exchange or dialyzing the samples before His-tag purification.

Expi293F™ cells must be recovered from freezing and passaged at least 3 times using the procedure outlined in the manual to ensure optimal performance. Cells should maintain performance for at least 30 passages if maintained in accordance with the protocol in the manual. 

While the Expi293™ Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F™ cultures beyond 5–6 x 10⁶ cells/mL, as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5–6  x 10⁶ cells/mL, passage them once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression Control IgG or an expression construct of known yield to determine if cell expression performance has been impacted.

The optimal expression time will be different for each protein, but most tested proteins fall within a 3–7 day window. As the system scales well, it is recommended you run a small-scale pilot experiment to determine when to harvest your protein of interest prior to scaling up.

The growth and expression characteristics of Expi293F™ cells are such that, by 7 days post-transfection, the culture medium should be close to being spent and maximal protein expression should have already been achieved. Continued incubation will result in a large decrease in cell culture viability.

The best transfection efficiencies are obtained when transfection complexes are used fresh. However, they should be stable for at least an hour. 

No. The Expi293™ Expression System is designed to run without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection.

This depends on the flask, but a general rule of thumb is to use one quarter the volume of the flask.

In the manual, we have provided optimal shake speeds for various formats. For most flasks, there would be a drop-off in expression when you go too fast or too slow because of cell shear stress or insufficient aeration. The effect is sharper in plates, where there is a sharper transition between static and moving fluid in wells. 

Our in-house scientists have cultured a 1 L volume in a 3 L baffled shake flask at 80–85 rpm. This was done using an Innova shaker with a 0.75-inch orbital throw.

We recommend cloning the heavy and light chain subunits separately into the pcDNA™3.4 TOPO® vector and then optimizing the ratios of the 2 plasmids. Each antibody needs to have the ratio of heavy to light chain adjusted for optimal antibody expression. Having the chains on separate plasmids makes this easier. Using the same plasmid will also ensure equal levels of expression of each subunit to start with. 

Please refer to the Application Note for tips on optimizing protein yield using the Expi293™ Expression System.

Yes, see the Application Note on using 96-well microtiter plates with the Expi293™ Expression System. Basically, you can use 700 μL in 2 mL block wells shaking at 1250–1500 rpm on a 3 mm throw vortex-type shaker, such as an Eppendorf® MixMate® mixer.

Please see the Application Note for a method for the adaptation of the Expi293™ Expression System for use in the WAVE Bioreactor™ System.

The two systems have the same components except for the transfection reagent and the medium supplied for formation of transfection complexes. The FreeStyle™ Max 293 Expression System contains FreeStyle™ Max Reagent and OptiPRO™ SFM (serum-free medium that is devoid of animal-origin components), whereas the FreeStyle™ 293 Expression System contains 293fectin™ Transfection Reagent and Opti-MEM® I Reduced Serum Medium (serum-free medium but not animal-origin free).

You should be able to use the ExpiFectamine™293 Reagent for transfection of Freestyle™ 293-F cells grown in FreeStyle™ 293 Expression Medium; however, the enhancers will provide only a little boost in expression as they are designed to work with higher density cultures that FreeStyle™ 293 Expression Medium cannot support. 

Both FreeStyle™ Max Reagent and 293fectin™ Transfection Reagent provide similar levels  of high transfection efficiency; however, FreeStyle™ Max Reagent has lower cytotoxicity and hence results in higher protein yields. Additionally, FreeStyle™ Max Reagent is animal-origin free.  

When comparing FreeStyle™ 293-F cells with FreeStyle™ CHO-S cells for average range of antibody and protein yield, it is definitely expected that antibody yield will be higher in FreeStyle™ 293-F cells than in FreeStyle™ CHO-S cells, when generated by transient transfection. On the other hand, antibody yields in stable CHO cells can be much higher. For non-antibody proteins, it is impossible to predict which of the two cell lines may provide higher yields.

Other 293 cell lines may be used with the FreeStyle™ 293 Expression System. However, they would have to be adapted to serum-free suspension culture in FreeStyle™ 293 Expression Medium and evaluated for transfection and expression.

With FreeStyle™ 293 Expression Medium, we would recommend harvesting the cells a little earlier to reduce any nonspecific binding by host cell proteins and then filtering the culture supernatant through a 0.2 or 0.1 µM membrane before loading onto the column. On the other hand, FreeStyle™ CHO Expression Medium contains EDTA that will strip the nickel column. The medium can be subjected to dialysis or buffer exchange prior to loading on the column, or the EDTA can be chelated by adding 1–3 mg/L NiSO4 or NiCl to the supernatant. Iron or cobalt salts can also be used.

Yes. Please visit the ExpiCHO page at thermofisher.com/expicho where you will find a short video showing exactly how expression with the ExpiCHO system is performed in our laboratories. This is an excellent resource for getting started or for troubleshooting.

Upon receipt of the cells on dry ice, it is best to either thaw the cells immediately or place the vials into liquid nitrogen storage for ~72 hours to allow cells to acclimate until the time of thaw; do not store cells at -80 degrees C. Once thawed and transferred into prewarmed media in a vented, non-baffled shake flask, cells should be incubated at 37 degrees C with 8% CO2 on a shaker platform set to 120 ± 5 rpm for a shaker with a 25 mm orbital diameter or 125 ± 5 rpm for a 19 mm orbital diameter. Cells should have high viability at the time of thaw and should recover quickly post-thaw, reaching their normal 18-hour doubling time within 1-2 passages.

Cells that are very clumpy or stringy in appearance or do not recover into a normal growth pattern within 1-2 passages may have been compromised during shipping or receiving, and shouldn't be used.

Within 1-2 passages post-thaw, ExpiCHO-S Cells should be growing with a doubling time of approximately 18 hours. There should be minimal cell clumping in the flasks, with only small clumps visible when the cells approach higher cell densities (i.e., ~6 x 10E6 cells/mL). When cells are cultured at 0.2-0.3 x 10E6 cells/mL, or 0.1-0.2 x 10E6 cells/mL, viable cell density should be approximately 4-6 x 10E6 cells/mL within 3 or 4 days, respectively. If cells are not growing within these approximate ranges, cell culture conditions will require further optimization.

In instances where ExpiCHO-S Cells are thawed and do not start to grow as noted above - attaining only relatively low densities in culture - one common solution is to verify the temperature of the cultures to ensure that the equipment settings are not generating too much heat. CHO cells are very tolerant to lower temperatures, but are more sensitive to elevated temperatures compared to HEK293 cells. Incubators, even when set to 37 degrees C, in conjunction with the heat generated from the shakers, can elevate the temperature in the culture flasks above optimal for CHO cells.

The optimal temperature of the media in the flask for ExpiCHO-S Cells should be around ~36.5 degrees C. If overheating is suspected, reduce the temperature of the incubator to reach optimal operating temperatures in the flasks. In such instances, it is best practice to thaw a new vial of cells rather than attempt to recover cells from elevated temperature conditions. Also, non-baffled flasks are recommended for use at all scales for both routine subculturing and protein expression runs.

ExpiCHO-S Cells do not reach log phase of growth until approximately 3 x 10E6 cells/mL cells, thus ExpiCHO-S Cells should be allowed to attain a density of 4-6 x 10E6 cells/mL at the time of subculturing to ensure the cells have reached log phase of growth. As cells approach 6 x 10E6 cells/mL, some very small cell clumps may be visible; do not try to break up the clumps, simply let them settle and remove the suspended cell solution for subculturing. For all cell manipulations, simply swirl flasks to resuspend the cells. Do not shake or pipet the cells vigorously to mix, as this can lead to decreased performance, especially just prior to transfection, when cells have attained very high densities.

ExpiCHO-S Cells should be passaged at least twice post-thaw and be growing within the ranges specified in the ExpiCHO system manual, prior to transfection. Cells should maintain consistent performance for at least 20 passages if maintained in accordance with the cell culture maintenance guidelines in the manual.

If cells significantly overgrow the target of 4-6 x 10E6 cells/mL (i.e., growing to 8-10 x 10E6 cells/mL), simply subculture the cells to a higher cell density than normal (i.e., seed new flasks at ~0.5 x 10E6 cells/mL) to allow the cells to recover. Since ExpiCHO-S Cells belong to a high-density cell line, it is not recommended to subculture the cells if they have not yet reached log phase of growth at 4-6 x 10E6 cells/mL, as this can impair growth over time.

As a quick check, ExpiCHO-S Cells can be seeded at 0.3 x 10E6 cells/mL in 30 mL of ExpiCHO medium in a 125 mL non-baffled flask, and the viability and viable cell density checked on days 5, 6, 7 postseeding. Typically, ExpiCHO-S Cells will reach maximal density around day 6 postseeding and should attain a density in the range of 20 x 10E6 cells/mL, and then will die off on day 7 and beyond. Determining the final viable cell density will be dependent upon the method used to count cells. Significant variability can be observed from different counting methods. If cells are exhibiting significantly different growth profiles, optimization of culture conditions should be performed. In such instances, it is typically useful to test multiple different shaking speeds simultaneously to determine which speed provides optimal cell growth and then start with this speed for your protein expression runs.

No. The ExpiCHO Expression System is able to achieve very high titers due to the way the components of the system have been optimized to work together for maximal protein expression. ExpiCHO medium is a transfection-compatible, high-density growth medium specifically matched to Gibco ExpiCHO Feed and ExpiFectamine CHO Enhancer. Other media are not compatible with the ExpiCHO system and may inhibit protein expression altogether.

ExpiCHO-S Cells should be subcultured at a density of 3-4 x 10E6 cells/mL one day prior to transfection to obtain a cell density of approximately 7-10 x 10E6 cells/mL on the day of transfection. These cells should be diluted to a final density of 6 x 10E6 cells/mL with fresh media and gently swirled to mix prior to transfection. Discard any remaining cells; do not reuse high-density cells for seeding of maintenance flasks.

For best results, it is recommended to perform DNA complexation in the following manner:

1. Dilute plasmid DNA into cold Gibco OptiPro medium.

2. At the time of use, dilute the ExpiFectamine CHO reagent with cold OptiPro medium and then immediately add to the diluted plasmid DNA.

3. Mix by gentle pipetting 1-2 times and/or inversion; do not vortex or pipet vigorously.

4. Complexation time should take 30 seconds - 5 minutes.

Avoid elongated hold times for the diluted ExpiFectamine CHO reagent or the ExpiFectamine CHO reagent-plasmid DNA complexation reaction.

If it is not possible to immediately add diluted ExpiFectamine CHO reagent to diluted plasmid DNA, we recommend diluting your plasmid DNA in the total complexation volume that would be used according to the kit protocol (i.e., the total volume of OptiPro media that would normally be used for diluting both the ExpiFectamine CHO reagent and the plasmid DNA) and then adding non-diluted ExpiFectamine CHO reagent directly to the diluted plasmid DNA. Mix by gentle pipetting 1-2 times and/or inversion. This method is useful for automation or small-scale transfections, where it is impossible or undesirable to add the ExpiFectamine CHO reagent to plasmid DNA immediately after dilution.

ExpiFectamine CHO is highly efficient for reagent transfection, enabling use of significantly lower levels of plasmid DNA for expression runs. Higher levels of DNA can be more stressful to the cells. The volume of ExpiFectamineCHO reagent specified in the kit protocol will account for using plasmid DNA in the range from 0.5 to 0.8 µg/mL; if using less DNA than this, the amount of ExpiFectamineCHO reagent should be reduced proportionately for best results. It is recommended to use 0.6-0.8 µg/mL for most proteins. For some proteins that are aggregate-prone or otherwise difficult to express, lower DNA levels may benefit expression.

For the majority of antibodies tested, a 1:1 ratio has been found to be ideal for the ExpiCHO system. The optimal heavy- to light-chain plasmid ratio is dependent upon the rate at which the two chains are expressed when cotransfected into the same cell, and may be antibody specific in some instances. We recommend starting with this ratio and then modifying as necessary for specific molecules. We recommend cloning the heavy- and light-chain subunits separately into the Invitrogen pcDNA3.4 TOPO vector initially, and then optimizing the ratios of the two plasmids. We have also tested single plasmids incorporating both heavy- and light-chain genes with comparable results, so in the end, the answer will come down to user experience and preference.

The ExpiFectamine CHO Transfection Reagent, ExpiFectamine CHO Enhancer, and ExpiCHO Feed are optimized to work together to provide maximal protein expression levels and are provided as a single kit for convenience. The ExpiFectamineCHO Transfection Reagent provides high transfection efficiency of high-density cultures, superior to any other transfection reagent. Expression levels greater than 30-fold higher are obtained using the ExpiCHO kit as directed versus substitution of polythyleneimine (PEI) for the transfection reagent, while using 50% less DNA in both methods.

We recommend using the pcDNA3.4 TOPO TA vector (Cat. No. A14697) for optimal expression. This vector contains the native, full-length cytomegalovirus (CMV) promoter and a woodchuck posttranscriptional regulatory element (WPRE) downstream of the cloning site. Other CMV-based vectors, however, have been shown to express similarly to the pcDNA3.4 TOPO TA vector, thus any CMV-driven vector is a good choice for the ExpiCHO system.

ExpiCHO Feed and ExpiFectamine CHO Enhancer should be added 18-22 hours posttransfection for best results. These solutions may be added to the flasks without prewarming. ExpiCHO Feed and ExpiFectamine CHO Enhancer may also be premixed ahead of addition to flasks to reduce the number of steps required. If using the standard protocol at 37 degrees C, it is recommended to add the feed and enhancer closer to the 18-hour timepoint.

When shifting the temperature for the high-titer and max-titer protocols, it is recommended that a dedicated 32 degrees C incubator be used, as simply changing the temperature of a 37 degrees C incubator to 32 degrees C may take a prolonged period of time to cool down and may limit the effectiveness of the temperature shift. Cooling the incubator by opening the door may result in contamination.

The time of harvest is highly dependent upon the nature of your protein. For stable proteins such as antibodies, we generally recommend harvesting on day 7-8 posttransfection (using the standard protocol), on day 9-10 (using the high-titer protocol, and on day 11-12 (using the max-titer protocol. Cell viability should still be high at this time, ideally 70-80% or greater. Intracellular proteins may require earlier harvesting, between days 4-7. For proteins that may be susceptible to degradation, a time course of harvesting should be considered to identify the optimal takedown time. In all instances, maintaining high cell viability at the time of harvest is ideal for both protein quality and purification.

No. The ExpiCHO™ Expression System is designed to run without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection, however there are an enhancer addition and 1-2 optional feed additions for improved yield.

Yes, you may be able to adapt your CHO cells into ExpiCHO™ medium.  Long-term adaptation in ExpiCHO™ medium may increase productivity of your CHO cells, and should sustain high-density growth.  However there is no guarantee that CHO lines other than Expi CHO-S™ cell line will achieve the same levels of expression as the ExpiCHO-S™ cells.  In limited testing, we have found other CHO subclones to be less easy to transfect than ExpiCHO-S™ cells in large-scale suspension format.

We do not recommend substituting another medium for ExpiCHO™ Expression Medium because it likely cannot support the same viable density as ExpiCHO™ Expression Medium, and may also contain components that inhibit the transfection using ExpiFectamine™ CHO.

Yes. For expression in 24- and 96-deep well blocks and 50 mL mini bioreactor tubes, please refer to the online protocol.

Our ExpiCHO-S™ Cells are derived from our cGMP banked CHO-S cells (Cat. No. A1155701), thus stable CHO-S selection methods are applicable to these cells.  For generation of stable CHO-S clones, however, we recommend to use the Freedom™ CHO-S™ (Cat. No. A1369601) or Freedom™ DG44 (Cat. No. A1373701) kits designed for this purpose.

With several test proteins in the ExpiCHO-S™ expression system, we see approximately 25-160 fold higher expression than FreeStyle™ CHO expression system and approximately 2-4 fold higher expression than Expi293™ expression system. Please see Figure 1 on the ExpiCHO™ web page at the following link for the data.

For both normal passaging and at the time of transfection for ExpiCHO-S™ cells, greater than 95% viability is critical for best results with the ExpiCHO™ expression system. We recommend for monitoring cell viability to use a trypan blue exclusion method (either manual or automated using a Vi-CELL Cell Viability Analyzer or Cedex Analyzer). Trypan blue stain is available for purchase (Cat. No. 15250061).

The formation of intact IgG molecules may be quantitated using a sandwich ELISA designed to capture and detect rabbit IgG. Besides the rabbit IgG positive control, reagents, and consumables that are included in the kit, you will also need purified rabbit IgG to be used as a standard, F(ab')2 goat anti-rabbit IgG HRP conjugate (Cat. No. A10547), Protein A-coated plates (Cat. No. 15130 for clear plates used in colorimetric detection), TMB colorimetric substrate (Cat. No. 34021), Antibody Dilution Buffer (Cat. No. 37535), and PBS or TBS buffer for washes.  There is an example procedure in our Protein A-coated plates manual. Please note, our R&D scientists determine titer values from crude cell culture supernatants using a Pall Life Sciences FortéBio™ Octet™ instrument equipped with a protein A biosensor.

For high-yield expression of functional membrane proteins in Expi293F™ cells, we offer the Expi293™ MembranePro™ Expression System (Cat. Nos. A25869, A25870) that combines the scalability and ease of use of Expi293™ and the technology of MembranePro™ to allow an increase of more than 20-fold in membrane protein yield compared to the standard, adherent culture MembranePro™ Functional Protein Expression System.

Note: Expi293F™ cells (Cat. No. A14527) and pEF6 V5 His TOPO® Expression Vector Kit (Cat. No. K961020) are not supplied with the Expi293™ MembranePro™ Expression System and have to be purchased separately as required.

No. However, you can visually tell if MembranePro™ particles formed via a pellet at the bottom of the tube following precipitation. You can also test the function of your MembranePro™ particles via receptor-ligand binding studies. 

While other expression vectors and promoters can be tested, we recommend using the pEF6 V5-His TOPO® TA expression vector, as the performance of the kit has been optimized for use with this expression vector containing the EF-1α promoter. 

While other cell lines can be tested, we recommend using the Expi293F™ cells, as the performance of the kit has been optimized for use with this cell line (i.e., transfection efficiency using ExpiFectamine™ Transfection Reagent). 

The GPCRs we produced and tested are ~50 kDa. In theory, there is no limit on the size of the extracellular or transmembrane domains. However, there may be packaging issues if the C-terminal domain is too large. 

Please refer to the Application Note for a protocol for adapting the MembranePro™ kit for use with the Expi293™ System.

The gag protein can't be removed unless you dissociate the MembranePro™ particles. Our recommendation is that methods used to isolate membrane proteins should eliminate gag. This system makes analytical quantities of protein, 50–500 μg total protein per reaction, of which a small fraction is your GPCR. 

Using G418 should not pose a problem for low-scale protein production for initial studies. However, we do not recommend adding G418 during large-scale protein production (i.e., in a bioreactor).

Even though the exact mechanism for MTX amplification is not well understood, the thinking is that both vectors tend to integrate in the same genomic locus and hence would get amplified at the same time.

We recommend cloning each protein subunit into each of the vectors to determine which vector combination gives the highest protein yield in transient transfection before making the stable cell line.