Insect Cell–Based Protein Expression

Thermo Fisher Scientific offers a variety of baculovirus systems (Table 3.1) to fit your needs:

• The Invitrogen BaculoDirect Baculovirus Expression System is a fast and easy method for generating recombinant baculovirus. BaculoDirect linear DNA includes attR sites for rapid and efficient recombinational cloning with an Invitrogen Gateway entry clone. The resulting recombinant DNA is taken directly from the Gateway LR reaction mix and used to transfect insect cells, saving a significant amount of time. Purified virus can be isolated within one week. The reduction of hands-on time makes the BaculoDirect system ideal for high-throughput expression.
• The Invitrogen Bac-to-Bac Baculovirus Expression System uses a unique bacmid shuttle vector that recombines by site-specific transposition and generates an expression bacmid in bacterial cells. The expression bacmid is then transfected into insect cells to generate recombinant baculovirus.
• The Invitrogen Bac-N-Blue Baculovirus Expression System has been used for more than a decade to produce high levels of recombinant proteins.
• The Drosophila expression system (DES) uses the well-characterized Drosophila Schneider S2 cells and simple expression vectors to allow stable or transient expression of recombinant proteins.
   

Table 3.1. Baculovirus-based expression systems offered and advantages of each.

Peak expression of protein in insect cells is dependent on several factors including: the multiplicity of infection (MOI), expression time, and the specific protein being expressed. Guidelines to optimize your system include using an MOI of 5–10 and an expression time of 48–72 hours. Protein expressed at times later than 72 hours may be processed aberrantly, because the large virus load can cause a breakdown of cellular processes.

Viral infection of insect cells will typically go through 3 stages that can be visually observed using an inverted phase microscope at 40x–400x magnification. The stages of viral infection (assuming high transfection efficiency) are:

Early

• Increased cell diameter—a 25–50% increase in the diameter of the cells may be observed
• Increased size of cell nuclei—the nuclei may appear to “fill” the cells

Late

• Cessation of cell growth—cells appear to stop growing when compared to a cells-only control
• Granular appearance
• Signs of viral budding—vesicular appearance of cells
• Viral occlusions—a few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell
• Detachment—cells release from the culture dish or flask

Very late

• A few cells may be filled with occluded virus, die, and burst, leaving signs of clearing in the monolayer

Once a viral stock has been created, titer determination of the stock is strongly recommended. A plaque assay can be performed to determine the titer of your viral stock. The main steps of performing a plaque assay are outlined below:

• Plate cells at 80% confluence in a 6-well plate
• Make a serial dilution of the P1 viral stock and add to cells
• Incubate for an hour at 27°C
• Mix 1% melted agarose into fresh medium
• Remove the viral supernatant
• Overlay the cells with the medium containing agarose
• Leave the plates for 2–3 hours for agar to completely solidify
• Incubate plates for 10–14 days
• Count plaques

When performing this assay, we suggest:

• Use cells that are in excellent health, of low passage (10–20), in log-phase growth, and high viability (>95%)
• Check viral stock for sterility (free of bacterial contamination)
• Use high-quality, low melting-point agarose
• The temperature of the medium with agarose is crucial—too hot, cells will die; but if too cold, it will solidify too quickly
• Wait 2–4 hours before removing the plate after overlay, so that the agarose can completely solidify
• Count plaques on a dilution plate

Use this equation to calculate the viral titer:
PFU/mL = number of plaques (PFU) / dilution factor x mL of inoculate

We suggest using a viral stock with a titer of ≥1 x 10⁸ PFU/mL for expression studies. To amplify your viral stock, infect cells at an MOI ranging from 0.05 to 0.1 (MOI is defined as the number of virus particles per cell). Note: If you have not determined the titer of your P1 viral stock, you may assume that the titer ranges from 1 x 10⁶ to 1 x 10⁷ PFU/mL.

Gibco insect media from Thermo Fisher Scientific has been formulated for maximum growth and protein yields. These media, in combination with Gibco pre-adapted cell lines (Table 3.2), provide a convenient system to save you time and effort.

See the insect media selection table

Insect cells are very sensitive to environmental factors. In addition to chemical and nutritional culture factors, physical factors can also affect insect cell growth; therefore optimization is required to maximize cell growth.

Consider the following when culturing insect cells:

• Temperature—the optimal range to grow and infect cultured insect cells is 27°C to 28°C
• pH—a range of 6.1 to 6.4 works well for most culture systems. Gibco Sf-900 II SFM will maintain a pH in this range under conditions of normal air and open-capped culture systems.
• Osmolality—the optimal osmolality of medium is typically 345 to 380 mOsm/kg depending on the cell line used
• Aeration—insect cells require passive oxygen diffusion for optimal growth and recombinant protein expression. Active or controlled oxygenated systems require dissolved oxygen at 10% to 50% of air saturation.
• Shear forces—suspension culture generates mechanical shear forces. Growing insect cells in serum-containing medium (10% to 20% FBS) generally provides adequate protection from cellular shear forces. If you are growing insect cells in serum-free conditions, supplementation with a shear-force protectant such as Gibco Pluronic F-68 Non-ionic Surfactant may be required. Note: Growing cells in Sf-900 II SFM or Gibco Sf-900 III SFM does not require addition of shear-force protectants.

For more information regarding insect cell culture, refer to the Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques.

Table 3.2. Summary of cell culture conditions for the most popular cell lines we carry.

BaculoDirect Baculovirus Expression System

The BaculoDirect Baculovirus Expression System uses a quick, 1-hour Gateway recombination reaction to produce the necessary bacmid for transfection (Figure 3.1), to help save days to produce recombinant baculovirus. Purified baculovirus can typically be isolated in less than 1 week. The main advantages of the BaculoDirect system include:

• Fast method generates recombinant virus in minimal time
• Strong polyhedrin promoter for high-level expression
• C-terminal or N-terminal 6xHis and V5 tag for easy detection and purification of recombinant proteins
• Flexible Gateway cloning allows use of any entry vector
• Thymidine kinase (TK) gene for negative selection of nonrecombinant virus using ganciclovir
• lacZ gene for quick determination of recombinant virus purity
• Simple, established protocol for high-throughput expression

Please also note that this DNA is linear, so the chance of generating nonrecombinant virus is minimized.

Our engineered BaculoDirect linear DNA contains attR sites for recombination of your gene of interest cloned into an Invitrogen Gateway Entry clone. Simply mix the entry clone with the BaculoDirect linear DNA and Invitrogen Gateway LR Clonase Enzyme, incubate for 1 hour, and then transfect either Sf9 or Sf21 insect cells to produce recombinant virus. We do not recommend using Invitrogen High Five Cells to generate viral stocks because of lower transfection efficiency. Once you have generated your high-titer viral stocks, you can use Sf9, Sf21, High Five, or Invitrogen Mimic Sf9 cells for protein expression. The need for transforming bacteria and isolating a large bacmid, or cotransfection of a transfer vector and linear baculovirus DNA into insect cells is eliminated. As a result, the hands-on time is greatly reduced and purified baculovirus can be isolated in 1 week.

The BaculoDirect linear DNA is designed for simple generation of recombinant baculovirus and expression in insect cells (Figure 3.2). In addition to attR sites for quick Gateway recombination cloning, the backbone contains a strong polyhedrin promoter for high protein expression and a C-terminal or N-terminal 6xHis and V5 tag for detection and purification.

Bac-to-Bac Baculovirus Expression System

The Bac-to-Bac Baculovirus Expression System relies on generation of recombinant baculovirus by site-specific transposition in E. coli rather than homologous recombination in insect cells to produce recombinant baculovirus. The expression cassette of the pFastBac vectors recombines with the parent bacmid in DH10Bac E. coli to form an expression bacmid. The parent bacmid contains the lacZ-alpha complementation factor for efficient blue/white screening of positive recombinants. The bacmid is then transfected into insect cells for production of recombinant baculovirus particles (Figure 3.3).

The main advantages of the Bac-to-Bac Expression Systems include:

• High titers (up to 10⁸ PFU/mL) enable large-scale protein expression
• pFastBac vector contains polyhedrin promoter for high yields of recombinant protein
• Reliable, rapid 5-minute blunt TOPO cloning helps save time
• N- and C-terminal 6xHis tag for easy purification
• TEV protease sites enable removal of N- or C-terminal tag, yielding a native protein

The pFastBac vectors offer the strong polyhedrin promoter for protein expression and a large multiple cloning site for simplified cloning. The Invitrogen Bac-to-Bac HBM TOPO Secreted Expression System enables secreted protein expression via the honeybee melittin (HBM) secretion signal, which is ideal for toxic proteins and glycoproteins that require a secretion signal to be glycosylated. Also, glycoproteins secreted from baculoviruses can be easily deglycosylated in vitro—an important feature for protein crystallization. With regard to packaging limit for the baculovirus, the baculovirus rod will continue to elongate as required to package the DNA. Thus, the system can theoretically accommodate hundreds of kilobases. Standard cloning techniques will limit the insert size before packaging limits become an issue.

Bac-N-Blue Transfection Kit

The Bac-N-Blue linear DNA found in the Invitrogen Bac-N-Blue Transfection Kit was specifically designed for recombination with the pBlueBac and pMelBac vectors. Recombinant viruses have a full-length, functional lacZ gene that results in the production of blue plaques. This allows for easy identification and purification. Bac-N-Blue linear DNA can be used with any polyhedrin promoter–based baculovirus transfer vector. The DNA is linearized at 3 sites, one of which is in a gene that is essential for viral propagation. This leads to a decrease in nonrecombinant virus, helping to make selection and purification of recombinant virus easy.

Drosophila Expression System (DES)

The DES system offers several advantages, including:

• Straightforward generation of insect cell lines that stably express high levels of your protein
• A variety of vectors with features such as an inducible promoter (metallothionein promoter) for expression of toxic proteins, secretion signals, and tags
• Easy high-density cell culture using Invitrogen Drosophila S2 Cells
• Nonlytic expression for reduced degradation

Stable S2 cell lines are generated by cotransfection of a DES expression vector with a selection vector, pCoBlast or pCoHygro (Figure 3.4). Once the expression construct is inside the S2 cell, hundreds of copies of the expression plasmid containing your gene of interest will spontaneously integrate into the genome. After a few weeks of selection, you can establish a polyclonal cell line that stably expresses high levels of your protein.