Search Thermo Fisher Scientific
Insect cells offer high levels of protein expression with posttranslational modification approaching that of mammalian cells, ease of scale-up, and simplified cell growth that can be readily adapted to high-density suspension culture for large-scale expression. Most of the posttranslational modification pathways present in mammalian systems also occur in insect cells, allowing the production of recombinant protein that is more antigenically, immunogenically, and functionally similar to the native mammalian protein than if expressed in yeast or other eukaryotes. Baculovirus expression systems are powerful and versatile delivery and expression vehicles for producing high levels of recombinant protein expression in insect cells. Expression levels up to 500 mg/L have been reported using the baculovirus expression system.
If you've found this chapter—Insect Cell–Based Protein Expression—useful, you may be interested in getting your own copy of the entire 118-page Protein Expression Handbook in convenient PDF format.
Thermo Fisher Scientific offers a variety of baculovirus systems (Table 3.1) to fit your needs:
System | Host | Secretion signal | Position | Purification | Epitope | Promoter | Expression /inducer | Advantage |
---|---|---|---|---|---|---|---|---|
Baculo-Direct system | Sf9, Sf21, or High Five Cells | – | N- or C-terminus | 6xHis | V5 | Polyhedrin | Infection | Fast and easy method for generation of recombinant baculovirus; ideal for high throughput |
Bac-to-Bac or Bac-to-Bac HBM system | Sf9, Sf21, or High Five Cells | Honeybee melittin | N-terminus | 6xHis | – | Polyhedrin or p10 | Infection production | Rapid baculovirus production; easy blue/ white selection of recombinant colonies |
Bac-N-Blue system | Sf9, Sf21, or High Five Cells | Honeybee melittin | C-terminus | 6xHis | Xpress V5 | Polyhedrin | Infection | Classic and trusted expression system for high-level recombinant protein production |
DES expression system | S2 cells | BIP | C-terminus | 6xHis | V5 | MT or Ac5 | CuSO4 or constitutive | Classic and trusted expression system for high-level recombinant protein production |
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:
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:
When performing this assay, we suggest:
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:
For more information regarding insect cell culture, refer to the Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques.
Temperature | Adherent culture | Suspension culture | Media with serum | SFM | Antibiotics | CO2-V | |
---|---|---|---|---|---|---|---|
Sf9 | 27°C ± 1°C | Yes | Yes | Grace’s supplemented (TNM-FH) with 10% heat-inactivated (HI) FBS; Add 0.1% Pluronic F-68 for suspension cultures | Sf-900 II SFM Sf-900 III SFM | Pen/Strep | No |
Mimic Sf9 | 27°C ± 1°C | Yes | Yes | Grace’s supplemented (TNM-FH) with 10% HI FBS; Add 0.1% Pluronic F-68 for suspension cultures | No | Pen/Strep | No |
Sf21 | 27°C ± 1°C | Yes | Yes | Grace’s supplemented (TNM-FH) with 10% HI FBS; Add 0.1% PluronicF-68 for suspension cultures | Sf-900 II SFM Sf-900 III SFM | Pen/Strep | No |
High Five | 27°C ± 1°C | Yes/No | Yes | No | Express Five SFM with the addition of glutamine | Pen/Strep | No |
S2 | 22°–24°C | Yes/No | Yes | Schneider’s media supplemented with HI FBS | Drosophila SFM | Pen/Strep | No |
D.Mel2 | 22°–24°C | Yes/No | Yes | No | Drosophila SFM | Pen/Strep | No |
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:
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.
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:
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.
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.
The DES system offers several advantages, including:
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.
Find more information on insect protein expression systems
For assistance with selecting the best vector for your experiment, try the Vector Selection Tool
For useful support resources, tips, and tricks related to getting started with your experiment, as well as troubleshooting help, go to the Protein Expression Support Center
仅供科研使用,不可用于诊断目的。