Mammalian Expression for High-Yield Protein Production Support—Getting Started
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For stable high-yield expression, we offer the Freedom® CHO-S® and Freedom® DG44 kits. For transient high-yield expression, we offer the 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.
High-Yield transient expression
All the components of the system are animal-origin free except for the Opti-MEM® I Reduced Serum Medium that is serum-free but not animal-origin free. Please see the Application Note for using the Expi293™ Expression System under animal origin–free conditions.
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.
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.
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.
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 Post-transcriptional Regulatory Element), both of which contribute to high level gene expression (about 2-3 fold higher expression than pcDNA™3.3 and 2-5 fold higher expression than with a standard pcDNA vector such as pcDNA™3.1.)
Our transfection reagent, enhancer, feed, and media are matched. They don’t work well with other reagents that we have tested. Use of transfection reagents other than the ExpiFectamine™ CHO Reagent to transfect high density ExpiCHO-S™ cultures can lead to substantially reduced performance. Expifectamine™ CHO with enhancer and feed provided a 30-fold yield improvement over ExpiCHO-S™ and ExpiCHO™ media with PEI for transfection when we tested expression levels with the rabbit IgG positive control.
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.
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.
High-Yield membrane protein production
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.
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.
High-Yield stable expression
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.
For Research Use Only. Not for use in diagnostic procedures.