Mammalian Cell–Based Protein Expression

Thermo Fisher Scientific offers the largest collection of mammalian expression vectors, specialized media, delivery reagents, and cells for efficient expression, selection, and analysis of recombinant proteins. There are many options for choosing a mammalian expression system matched to specific needs. The main criteria to be considered for selection are:

What delivery method will work best for you?

You can introduce your gene of interest into mammalian cells by transfecting the plasmid DNA using chemical reagents, by electroporation, or by viral transduction using recombinant viruses. The choice of delivery method depends upon the mammalian cell type that is being used and the experimental effects desired.

Transfection works well for a wide variety of cell types, whereas electroporation using the Invitrogen Neon Transfection System or viral transduction is the ideal choice for cell types that are difficult to transfect or for nondividing cell types.

• For viral transduction of the gene of interest, you can choose from the Invitrogen ViraPower lentiviral and adenoviral expression systems.
• If transfection is the delivery method of choice, consider the further points shown below:

Will you be expressing the protein transiently or making stable cell lines?

Mammalian expression experiments are performed either transiently or with stable cell lines that express the protein of interest.

• Transient expression, which typically results in high levels of expression for a few days, is ideal for rapid protein production and quick data generation. Any mammalian expression vector can be used for this purpose.
• Stable expression requires the generation of stable cell lines in which the expression construct is integrated into the host genome. These stable cell lines can be used over a long experimental time course or used over many experiments. Because the expression vector has a selection marker (such as an antibiotic resistance gene), cells that have integrated the construct can be selected by the addition of a selection agent (such as the antibiotic) to the medium (Table 2.1). In order to determine the optimal antibiotic concentration to use when establishing a stable cell line, we recommend performing a dose-response curve or kill curve:
  • Plate untransfected cells at 25% confluence and grow them in medium containing increasing concentrations of the antibiotic. For some antibiotics, you will need to calculate the amount of active drug to control for lot variation.
  • Replenish the selective medium every 3–4 days. After 10–12 days, examine the dishes for viable cells. The cells may divide once or twice in the selective medium before cell death begins to occur.
  • Look for the minimum concentration of antibiotic that results in complete cell death. This is the optimal antibiotic concentration to use for stable selection.
     

Table 2.1. Eukaryotic selection antibiotics.

For random integration of the expression construct, any of our Invitrogen pcDNA vectors may be used. For targeted integration of the expression construct, we offer the Invitrogen Jump-In System and Invitrogen Flp-In System.

Do you need inducible or constitutive expression?

• An inducible promoter allows you to control the timing of gene expression. In the absence of the inducer, the gene is not expressed. Addition of the inducer turns on expression. This option is ideal for expressing toxic proteins. For regulated and inducible expression of the gene of interest, we offer the Invitrogen T-REx Expression System, Invitrogen Flp-In T-REx system, and the Invitrogen GeneSwitch System (Table 2.2).
   

Table 2.2. Mammalian cell–based expression systems compared.

• On the other hand, if you are working with a nontoxic gene and the timing of expression is not important, choose an expression vector with a constitutively expressing promoter. For this purpose, we offer the pcDNA vectors. There are several options to choose from, depending upon your cell type. pcDNA vectors are available with either the CMV, EF-1, or UbC promoter, a variety of different epitope tags, standardized detection or purification across a range of proteins, several selection markers for creating stable cell lines, and different cloning formats, such as restriction enzyme cloning, TOPO cloning, and Gateway cloning.
• We also offer a wide selection of specialized mammalian expression vectors designed for specific applications, including episomal expression, secreted expression, and intracellular targeting.

This handbook will highlight the main mammalian expression vectors and systems we offer for each of the expression categories described below.

Mammalian transient expression systems enable flexible and rapid production of proteins (Figure 2.1). They are ideal for generating large amounts of protein within 1 to 2 weeks. We offer two fully optimized, mammalian transient expression systems for ultrahigh protein yields in CHO and HEK 293 cells. These systems enable expression of human or other mammalian proteins with more native folding and posttranslational modifications—including glycosylation—than expression systems based on hosts such as E. coli, yeast, or insect cells.

The Gibco ExpiCHO mammalian transient expression system marks a revolutionary leap forward in the transient production of recombinant proteins in CHO cells. This fully optimized system has been designed to deliver protein yields up to 3 grams per liter, which is higher than the best HEK 293–based systems. The ExpiCHO system enables you to rapidly and cost-effectively access CHO cell–expressed proteins early in the drug development process, providing you the highest confidence that transiently expressed drug candidates will mimic downstream biotherapeutics manufactured in CHO.

The Gibco ExpiCHO Expression System brings together a high-expressing CHO cell line, a chemically defined animal origin–free culture medium, an optimized culture feed, and a high-efficiency transfection reagent that synergistically act to provide titers as much as 160x higher than the Gibco FreeStyle CHO Expression System and 3x higher than the Gibco Expi293 Expression System. Expression levels of up to 3 g/L were achieved for human IgG proteins (Figure 2.2).

The ExpiCHO Expression System will revolutionize the use of CHO cells for transient protein expression during early phase drug candidate screening. The glycosylation patterns of recombinant IgG produced by the Expi293 and ExpiCHO transient expression systems were compared to the same protein expressed in stable CHO cells. It is clear that glycosylation of recombinant IgG produced in the ExpiCHO system is much more like glycosylation of the stable CHO cell system (Figure 2.3) which provides a very strong correlation between transiently expressed drug candidates and downstream biotherapeutics manufactured in CHO.

The ExpiCHO Expression System provides simple and flexible protocols designed to achieve the perfect balance of protein yields, speed, and scalability to meet your specific needs or application (Figures 2.4–2.6).

The Expi293 Expression System is a major advance in transient expression technology for rapid and ultrahigh-yield protein production in human cells. It is based on high-density culture of Gibco Expi293 Cells in Gibco Expi293 Expression Medium. Transient expression is powered by the cationic lipidbased Gibco ExpiFectamine 293 transfection reagent in combination with optimized transfection enhancers designed to work specifically with this transfection reagent. All components work in concert to generate 2- to 10-fold higher protein yields than are attained with previous 293-transient expression systems. Expression levels of greater than 1 g/L can be achieved for IgG and non-IgG proteins (Figure 2.7).

Expi293 Expression Medium is a chemically defined, serum-free, protein-free medium that enables high-density cell culture and transfection, allowing for significantly higher volumetric protein yields than traditional culture systems (Figure 2.8).

Expi293F Cells are specifically adapted to achieve higher pg/cell/day productivity than typical HEK 293 cells, and produce up to 1.7 times more protein per cell than Invitrogen Freestyle 293-F Cells when used in the Expi293 Expression System. ExpiFectamine 293 transfection reagent is capable of high-efficiency transfection of high-density HEK 293 cultures, and is coupled with transfection enhancers that further boost transfection performance and expression levels (Figure 2.9).

The Expi293 Expression System is highly scalable and should produce similar volumetric protein yields in transfection formats ranging from 1 mL cultures in a 24-well plate up to 1 L cultures in shaker flasks (Figure 2.10), and we have application notes to guide your use of 10 L bioreactor cultures.

The Gibco Expi293 MembranePro Expression System combines the benefits of the Expi293 Expression System and the Gibco MembranePro Expression System. It is designed for high-yield transient expression and display of mammalian cell surface membrane proteins, including G-protein– coupled receptors (GPCRs), in an aqueous-soluble format.

The Expi293 MembranePro Expression System generates virus-like particles (VLPs) to capture lipid raft regions of the cell’s plasma membrane as the VLPs are produced and released from the surface of the cell. Using this system, it is possible to capture and display endogenous or overexpressed GPCRs and other cell-surface membrane proteins in their native context for downstream assays. Because the VLPs are packaged by the cell and secreted into the culture medium, they allow the isolation of functional membrane proteins by simply decanting and clarifying the culture medium, and isolating the VLPs by precipitation. This represents a substantial savings in time, effort, and required machinery over preparing cell membrane fractions. Because VLPs capture receptor rich regions of the plasma membrane, your GPCR may also be substantially enriched over crude membrane preparations.

The Expi293 MembranePro Expression System provides a greater than 20-fold increase in membrane protein yield compared to the adherent-cell MembranePro system (Figure 2.11). Other benefits include ease of use and scalability.

We offer 2 CHO-based Gibco Freedom cell line development kits: Gibco Freedom CHO-S Kit and Gibco Freedom DG44 Kit. With these kits, commercial licensing is simple, flexible, and without royalties. Both kits offer completely integrated workflows using animal origin–free reagents and fully documented parental cell lines for consistent generation of IgG-producing scalable clones that meet current industry standards for productivity. The kits are easy to use and can take your gene of interest from transfection to lead clone, typically in less than 6 months.

Jump-In cell engineering platform

The Invitrogen Jump-In targeted integration technology uses PhiC31 integrase–mediated recombination to stably integrate your choice of DNA sequence at specific genomic locations, called pseudo-attP sites, in mammalian cells. Unlike the better-known recombinases, such as Cre and Flp, PhiC31 integrase catalyzes recombination between 2 nonidentical sites and, combined with the lack of a corresponding excisionase enzyme, makes the integration events unidirectional and irreversible.

Jump-In Fast Gateway System

The Invitrogen Jump-In Fast Gateway System combines the Invitrogen MultiSite Gateway Pro cloning and Invitrogen Jump-In cell engineering technologies, to allow generation of a polyclonal pool of well-expressing stable clones in just 2 weeks. The system uses PhiC31 integrase to integrate the genetic material into the cell background, to produce a pool of retargeted cells with a high proportion of positives. You need to screen only 10 clones to find one with your ideal expression level.

In the Jump-In Fast Gateway System, the integrated DNA sequences include your genetic elements of interest (such as promoter-reporter pairs) from the targeting expression construct that is generated using the pJTI Fast DEST vector and the Invitrogen MultiSite Gateway Pro Plus Vector Module. Because the pJTI Fast DEST vector also carries the hygromycin-resistance gene, transformants containing stably integrated sequences can be selected using hygromycin B and expanded for downstream applications (Figure 2.12).

Jump-In TI Gateway System

The Invitrogen Jump-In TI Gateway System combines the MultiSite Gateway Pro cloning and Jump-In cell engineering technologies to enable the creation of isogenic, stable cell lines that express your gene of interest. The system initially uses R4 integrase to generate a platform cell line containing the targetable R4 attP sites in the preferred cell background, after which PhiC31 integrase is used for retargeting the expression construct that contains the genetic elements of interest (Figure 2.13). The Jump-In TI Gateway System enables elimination of chromosomal positioning effects from your experiments.

Jump-In parental cell line kits

Invitrogen Jump-In parental cell line kits allow you to generate isogenic cell lines by providing a parental (platform) cell line with a single R4 attP site ready to be retargeted with your genetic material.

Flp-In system

The Invitrogen Flp-In system allows for stable integration and expression of your gene of interest to deliver single-copy isogenic cell lines. Flp-In expression involves introduction of a Flp recombination target (FRT) site into the genome of your chosen mammalian cell line. An expression vector containing your gene of interest is then integrated into the genome via Flp recombinase–mediated DNA recombination at the FRT site (Figure 2.14).

We offer a selection of Flp-In products, including expression vectors and systems, as well as parental cell lines with a stably integrated FRT site. Designed for rapid generation of stable cell lines, these products enable rapid, reproducible, high-efficiency integration and high-level expression of your protein of interest from a Flp-In expression vector.

Flp-In T-REx system

The Flp-In T-REx system combines the targeted integration of the Flp‑In system with the powerful inducible expression of the T-REx system to allow rapid generation of isogenic, stable mammalian cell lines exhibiting tetracycline-inducible expression of a gene of interest from a specific genomic location (Figure 2.15). We also offer the Invitrogen Flp-In T-REx 293 Cell Line, designed to save you time in expression experiments.

For researchers studying G-protein–coupled receptors (GPCRs) and other membrane proteins, MembranePro expression systems deliver enriched, functional membrane proteins efficiently and reliably.

• Proteins are displayed on mammalian cell membranes
• Cellular quality control and mammalian posttranslational processing help produce functional proteins
• Proteins bud off on lipoparticles and are easily collected from the culture medium
• Lipoparticles are enriched with expressed receptors

The Invitrogen MembranePro Functional Protein Expression System is available for adherent cell cultures, and the Expi293 MembranePro Expression System is available for suspension cultures.

The MembranePro Functional Protein Expression System is designed for efficient expression and display of functional mammalian cell-surface membrane proteins, including G-protein–coupled receptors (GPCRs), in an aqueous-soluble format. The system uses virus-like particles (VLPs) to capture lipid raft regions of the cell’s plasma membrane as the VLPs are secreted from the cell. Using this system, it is possible to capture and display endogenous or overexpressed GPCRs and other cell-surface membrane proteins in their native context for downstream assays. Because the VLPs are packaged by the cell and secreted into the culture medium, they allow the isolation of functional membrane proteins by simply decanting and clarifying the culture medium, and isolating the VLPs by precipitation. This represents a substantial savings in time, effort, and required machinery over preparing cell membrane fractions. Because VLPs capture receptor-rich regions of the plasma membrane, your GPCR may also be substantially enriched over crude membrane preparations.

The MembranePro Functional Protein Expression System takes advantage of the functionality of the lentiviral gag protein, which, when expressed in 293FT cells, travels to the plasma membrane where it forms buds underneath the lipid rafts. Because lipid rafts play an active role in regulating the conformational state and dynamic sorting of membrane proteins, recombinant and endogenous GPCRs and other receptors are localized in these microdomains after having passed the cells’ “quality control”. As the VLP buds from the cell, it becomes enveloped in this portion of the plasma membrane and captures the membrane proteins in their native context.

By capturing just the membrane protein–rich lipid rafts, this versatile and ready-to-use system distinguishes itself from crude membrane fractions, which contain total plasma membrane, as well as contaminating amounts of endoplasmic reticulum, Golgi apparatus, and nuclear envelope.

The Expi293 MembranePro Expression System combines the benefits of the Expi293 Expression System and the MembranePro expression system. It is designed for high-yield transient expression and display of mammalian cell surface membrane proteins, including GPCRs, in an aqueous-soluble format.

If you are using a hard-to-transfect mammalian cell line, an animal model, or need higher-efficiency gene delivery, our Invitrogen ViraPower product portfolio provides a good option for either transient or stable expression. We offer ViraPower systems for lentiviral expression and adenoviral expression (Table 2.3). Both systems are highly efficient and create replicationincompetent viral particles to help enable safe, effective, reproducible delivery and expression of your gene in any mammalian cell type.

Table 2.3. Adenovirus and lentivirus delivery systems compared.

Invitrogen ViraPower lentiviral expression systems stably integrate your gene of interest into your target cell’s genome, making these systems ideal for functional studies. They provide efficient viral delivery and long-term gene expression in both dividing and nondividing cells (e.g., stem cells).

The Invitrogen ViraPower HiPerform lentiviral expression kits achieve elevated protein expression (4-fold higher expression than standard lentiviral kits), even in nondividing cells such as stem cells and primary neuronal cells, due to the presence of woodchuck hepatitis virus posttranscriptional regulatory enhancer (WPRE) and central polypurine tract (cPPT) elements in the lentiviral expression vector. The vectors are available in Gateway and TOPO cloning formats to enable flexible and efficient cloning. The Invitrogen ViraPower HiPerform lentiviral FastTiter kits enable accurate determination of functional lentivirus titers in just 2 days, using emerald green fluorescent protein (EmGFP) as a reporter.

We also offer standard ViraPower lentiviral expression kits, in which the lentiviral expression vector lacks the WPRE and cPPT elements.

The Invitrogen ViraPower adenoviral expression system enables reliable gene delivery and high-level transient gene expression from the CMV promoter or from a promoter of your choice. Use of this system allows the creation of replication incompetent (E1 and E3 deleted) adenoviral particles that deliver and express high levels of the gene of interest in dividing and nondividing cells. The adenoviral expression vector is Gateway system–adapted to enable fast, easy, and accurate cloning of the gene of interest.