Insect Expression Support—Getting Started
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Yes, baculovirus is a good candidate for the problem of expressing toxic proteins (i.e., membrane proteins). The polyhedron promoter does not express at maximal levels until 18–24 hr after infection. The polyhedron promoter is active late in the lytic cycle. That being said, it is minimally active as early as 8 hours, so if the gene is very toxic, there may be a problem. The solution in that case would be to switch to an inducible expression system. Transmembrane proteins can often be difficult to express in any system.
Peak expression of protein in insect cells is dependent on the multiplicity of infection (MOI), expression time, and the 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.
GOI Cloned in
Bac-N-Blue™ baculovirus DNA
Homologous recombination (N-term: lacZ; C-term: essential ORF 1629 sequence)
LacZ and ORF1629 sequence with 3’ LacZ and 5’ ORF1629
Bacmid containing baculovirus DNA
Transposition of the GOI from donor vector into the bacmid containing baculovirus DNA
miniTn7R/L with respective counterpart target sequences in bacmid
BaculoDirect™ linear baculovirus DNA
Recombination through LR reaction
attL1/L2 with attR1/R2
Recombination Happens in
How to Select for Positive Recombination
Plaque assay and PCR verification of the viral DNA
The linear baculovirus DNA is compatible with other companies’ vectors
DH10Bac E. coli cells
Blue/white screening of E. coli DH10Bac cells and PCR verification of the bacmid
No plaque purification needed; only positive recombination DNA is transfected
PCR verification of the LR reaction and ganciclovir negative selection
Low P1 viral titer
Please follow the recommendations below:
- Cells should be in excellent health, of their low passages (5–15), in log-phase growth, with viability >95%
- DNA must be of high purity, free of endotoxin
- No antibiotics should be used during transfection
- Cellfectin® reagent has to be completely resuspended
- Include controls (media control, DNA control, and transfection reagent control) for comparison and troubleshooting
Adherent Sf9 cells round up and show a smaller contact point. Infected Sf9 cells in suspension culture round up and look larger when infected.
Yes, we offer our Bac-to-Bac® HBM TOPO® Secreted Expression System (Cat. No. A11338 or A11339), which uses the honeybee melittin (HBM) secretion signal. This system is ideal for the study of toxic proteins and glycoproteins.
Please see the description below of the different stages of viral infection:
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.
Cessation of cell growth—cells appear to stop growing when compared to a cell-only control.
Signs of viral budding—vesicular appearance of cells.
Viral occlusions—few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
Detachment—cells release from the dish or flask.
Very late cell lysis—a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.
Yes. Contamination of your recombinant DNA with uncut (occ+) DNA will lead to dilution of your recombinant virus over time because, in general, uncut (wild-type, occ+) virus infects and replicates at higher efficiency than recombinant virus. Also, initiating expression studies with a pure, single virus population will ensure reproducible results.
If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4°C, and protect from light. Aliquots can be stored at –80°C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.
Yes, large-scale expression experiments can be performed. Please see the table below for different large-scale methods, requirements, added benefits, and references:
Bac-to-Bac® Baculovirus Expression System
The MOI, or multiplicity of infection, is the average number of viral particles that infect a single cell in a specific experiment. You can calculate the MOI with the following equation:
MOI (pfu/cell) = [titer (pfu) x viral stock volume (mL) used in inocula] / [cell density (cells/mL) x culture volume (mL)]
Yes, it is possible. Several five-subunit proteins, such as human replication factor C, have been expressed using recombinant baculovirus. We recommend that a separate high-titer stock (HTS) of each subunit be produced to optimally express the multi-subunit protein. This way, the amount of each subunit expressed can be controlled by varying the multiplicity of infection (MOI) of each subunit's HTS. Please refer to the following articles for more information:
- Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192–8197.
- Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355–9358.
- Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16–22.
While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:
- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
Biochim Biophys Acta 1260(1):14–20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149–153.
The promoter that drives the gene of interest is the polyhedron promoter. This promoter can be substituted by the p10 promoter, though the polyhedron promoter is generally 3–5 times stronger for most proteins. However, a protein that is highly modified or secreted is often expressed much more efficiently by the p10 promoter, as it becomes active in very early late phase, as opposed to the polyhedron promoter, which is not active until very late phase. Cellular protein synthesis that is required for the efficient and correct processing of complex proteins is shut down during the very late phase. This explains why some reports mention the expression of secreted and modified proteins where the p10 promoter is just as efficient as the polyhedron promoter or as much as 2x higher than the polyhedron promoter. In most cases, however, the polyhedron promoter is working just fine. The determination as to which is the stronger promoter will depend on a number of factors, including the nature of the protein and the time of harvest post-infection.
The polyhedron protein is 30 kDa.
Our R&D team has successfully expressed proteins up to 300 kDa. If they express in >2% serum, it should minimize degradation. If you don’t mind the extra step of purification, 10% serum could be used. We highly recommend doing a time-course infection with high-titer stock, with a MOI of 5–10, to make an assessment of the minimum harvesting time necessary for the best expression. Time points should be taken every 24 hours for 5 days.
Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:
- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754–16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177–183.
The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.
The following is an excellent reference for how to prevent proteolytic artifacts in the baculovirus expression system:
Hom LG, Volkman LE (1998) Preventing proteolytic artifacts in the Baculovirus expression system. BioTechniques 25:18–20.
5’ sequencing primer: 5’ – TAT TCC GGA TTA TTC ATA CC – 3’
3’ sequencing primer: 5’ – TTC AGG TTC AGG GGG AGG TG – 3’
The expected PCR size will depend on the pFastBac™ vector that got transposed into the bacmid:
- Bacmid alone: 300 bp
- With pFastBac™ 1: 2300 bp + size of insert
- With pFastBac™ 1-Gus: 4200 bp
- With pFastBac™ HT: 2430 bp + size of insert
- With pFastBac™ HT-CAT: 3075 bp
- With pFastBac™ Dual: 2560 bp + size of insert
- With pFastBac™ Dual-Gus/CAT: 5340 bp
- With pDEST8: 2316 bp + size of insert
- With pDEST10: 2484 bp + size of insert
- With pDEST20: 3031 bp + size of insert
BaculoDirect™ Baculovirus Expression System
Begin your BaculoDirect™ experiments by cloning in your gene of interest into your Gateway® Entry Vector, followed by the LR Clonase® reaction into the BaculoDirect™ vector. Transfect this vector into your cells and grow for 3 days. On the 4th day, collect P1 viral stock. Re-infect cells, and grow for 3 days. On the 7th day, collect P2 viral stock. Infect cells, followed by harvesting of protein and purification on the 10th day.
We recommend using Sf9 or Sf21 cells to generate high-titer viral stocks. We do not recommend using High Five™ cells to generate viral stocks due to lower transfection efficiency. Once you have generated your high-titer viral stocks, you can use Sf9, Sf21, High Five™, or Mimic™ Sf9 cells for protein expression.
The TK gene is for negative selection of non-recombinant virus using ganciclovir.
Proteins have been efficiently secreted utilizing baculovirus signal sequences. Please see the following references:
- Kuhn S, Zipfel PF (1995) The baculovirus expression vector pBSV-8His directs secretion of histidine-tagged proteins. Gene 162:225–229.
- Krol BJ, Murad S, Walker LC, Marshall MK, Clark WL, Pinnell SR, Yeowell HN (1996) The expression of a functional, secreted human lysyl hydroxylase in a baculovirus system. J Invest Dermatol 106:11–16.
Investigators have been successful with the honeybee melittin secretion sequence. Please see the following references:
- Tessier DC, Thomas DY, Khouri HE, Laliberté F, Vernet T (1991) Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide. Gene 98:177–183.
- Garnier L, Cahoreau C, Devauchelle G, Cérutti M (1995) The intracellular domain of the rabbit prolactin receptor is able to promote the secretion of a passenger protein via an unusual secretory pathway in lepidopteran cells. BioTechnology 13:1101–1104.
- Vihko P, Kurkela R, Porvari K, Herrala A, Lindfors A, Lindqvist Y, Schneider G (1993) Rat acid phosphatase: Overexpression of active, secreted enzyme by recombinant baculovirus-infected insect cells, molecular properties, and crystallization. Proc Natl Acad Sci U S A 90:799–803.
- Mroczkowski BS, Huvar A, Lernhardt W, Misono K, Nielson K, Scott B (1994) Secretion of thermostable DNA polymerase using a novel baculovirus vector. J Biol Chem 269:13522–13528.
Bac-N-Blue™ Expression System
In the Bac-N-Blue™ system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue™ vector is a linearized AcMNPV derivative that contains an incomplete (3’) lacZ fragment. The corresponding transfer vector contains a 5’ lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue™ baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue™ baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac® expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac® expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 μg/mL kanamycin (bacmid), 7 μg/mL gentamycin (pFastBac™), 10 μg/mL tetracycline (helper plasmid), 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.
“Wild-type” refers to uncut Bac-N-Blue™ DNA that will produce occlusion body positive (occ+) plaques. The frequency of wild-type plaques is less than 20%.
Yes, any baculovirus transfer vector can be used so long as it contains ORF603 and ORF1629 sequences.
We recommend designing the forward and reverse primers with the following sequences:
Forward primer: 5´-TTTACTGTTTTCGTAACAGTTTTG-3´ Tm = 62°C
Reverse primer: 5´-CAACAACGCACAGAATCTAGC-3´ Tm = 58°C
These primers flank the polyhedron region and are compatible with all polyhedron promoter–based baculovirus transfer vectors. The forward PCR primer binds from –44 (nt 4049) to –21 (nt 4072) in front of the start of the polyhedron gene, using the nomenclature of O'Reilly et al., 1992. The reverse PCR primer binds at +794 (nt 4886) to +774 (nt 4866) 3´ of the polyhedron gene.
Plaque Assay and Viral Stocks
We recommend you perform a plaque assay to determine the titer of your viral stock. You may also perform a plaque assay to purify a single viral clone, if desired.
Please see the method below for an outline of the main steps of performing a plaque assay:
- Plate cells at 80% confluency in a 6-well plate
- Make a serial dilution of the P1 viral stock (1–10-5) and add to cells
- Incubate for an hour at 27°C
- Mix 1% melted agarose into the 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 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 100% solidify
- Count plaques on a dilution plate
(1/dilution) x # of plaques = pfu/mL
e.g., if you have 50 plaques on the 10-6 plate, then you have 1(10-6) x 50 = 5 x 107 pfu/mL
Please see the equation below:
pfu/mL = number of plaques (pfu)/dilution factor x mL of inocula
So, if you have a well with viral dilution of 10-8 containing 18 white plaques, the viral titer is calculated as followed:
X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 109 pfu/mL
We suggest using a viral stock with a titer of >1 x 108 pfu/mL for expression studies.
You can stain the monolayer with neutral red or MTT to make the plaques more visible. Alternatively, you can allow the plates to develop for a few days longer (2–5 days on average) at room temperature to increase the contrast in recombinant plaques. However, the plaques stained with neutral red cannot be used for plaque purification and viral amplification.
When propagating virus stock, use a low MOI (0.03–0.1) in order to avoid effects of defective interfering particles (DIPs). A low MOI, which ensures no more than 1 virion per cell, prevents the amplification of DIPs. A harvest time based on 15% cell viability is appropriate.
NOTE: DIPs are nearly normal virus capsids containing genomes that are defective and are unable to undergo successful replication. While this "particle" is not infectious by itself, it can replicate when co-infected with normal virion, or with some other types of DI particles.
This is dependent on how much virus is added. If cells are infected at an MOI of 5, usually cells are infected at 24 hours, and cells begin to lyse at around 65 hours. If less virus is used, this takes longer, and more virus takes less time.
We recommend harvesting high-titer virus when there is 90% cell lysis. This takes approximately 5–7 days. If the cells go longer, the proteases released from the lysed cells will start to degrade viral surface proteins and result in less infectious virus.
Our R&D team will typically pick a plug and add it to a 12-well dish with 0.5 x 106 cells/well and 2.5 mL total volume per well. After approximately 3 days, remove 0.75 mL to make DNA for PCR and keep the remaining medium in an Eppendorf tube as your P1 viral stock. As an aside, it is okay to pick a plaque and store it in Grace’s medium.
Typically, 0.5 x 106 cells per well in 2.5–3 mL is a good starting point. Lysis should begin by day 3. Virus may be harvested and amplified between 3 and 7 days (90% cell death).
Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks.
Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.
Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don’t recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.
If this lower-titer stock is a P1 or P2 stock, a viral amplification protocol can be used. If the low-titer stock was once a high-titer stock, but has dropped titer due to age or the stock was propagated many generations, then it may be necessary to regenerate the high-titer stock. If the high titer stock is >P5, then there may be an excessive amount of defective interfering particles that infect cells but do not properly replicate or produce protein. If the existing stock is plated out and a fresh plaque is re-isolated (DIPs do not form plaques), a new high-titer stock can be established.
ExpiSf Expression System
The easiest way to get started is by purchasing the ExpiSf Expression System Starter Kit (Cat. No. A38841). This is an all-in-one kit that contains all of the reagents necessary to generate recombinant baculovirus and express your proteins. Reagents for cloning your gene of interest into the pFastBac Expression Vector are sold separately and can easily be found here.
Yes. There is a system page at www.thermofisher.com/expisf with comprehensive product information. We also offer our ExpiSf LabCoat Live virtual training course that provides interactive online lectures. For a thorough understanding of the system, experiment setup, and data analysis, sign up here (check for availability in your region). These are excellent resources for getting started or for troubleshooting.
We offer LabCoat Live: SmartStart Training for the ExpiSf Expression System. LabCoat Live is a unique learning environment that allows you to gain hands-on experience in your lab by providing the necessary reagents and protocols, and offering lectures through a flexible and affordable online environment with a self-paced lab experiment.
No. The ExpiSf Expression System is able to achieve high protein yields because of the way the components of the system have been optimized to work together for maximal baculovirus production and protein expression. ExpiSf CD Medium is a chemically defined, yeastolate-free, high-density growth medium specifically matched to ExpiSf Enhancer. ExpiSf CD Medium is essential to support high-density growth of ExpiSf9 Cells and helps enable high-density infections.
We always recommend titering your baculovirus stock prior to protein expression experiments. A fast and easy-to-follow FACS-based method for baculovirus quantification can be found in the ExpiSf Expression System User Guide. However, if you choose not to titer your virus stock, we recommend using 250-500 µL of P0 virus stock for protein expression at the 25 mL culture scale (1:100 to 1:50 dilution). For optimal protein expression, we recommend performing a titration of your virus to determine the optimal amount to be used in your infections.
Baculovirus stocks can be stored at 4 degrees C protected from light for up to 12 weeks. Alternatively, they can be stored at -80 degrees C or in liquid nitrogen for longer periods. We do not recommend repeated freeze/thaw cycles of your virus stock. Frozen virus should be stored in small aliquots and not re-frozen once thawed. Following these guidelines, baculovirus stocks can be stored without DMSO or cryopreservatives (e.g., FBS or BSA). In instances of unstable virions, 0.1% - 1% BSA may be added to stabilize the virus. Store the virus stocks in polypropylene containers or siliconized glassware to prevent non-specific binding of virus. They should be re-titered periodically if used as inoculates.
We recommend using the Bac-to-Bac Baculovirus Expression System with the ExpiSf Expression System. We have not tested system performance using other baculovirus expression vector systems (BEVS). If using other BEVS, experimental conditions will have to be established empirically.
Upon receipt of ExpiSf9 Cells on dry ice, our recommendation is to either thaw the cells immediately or place the vials in liquid nitrogen (vapor phase) storage for ˜72 hours to allow cells to acclimate until the time of thaw. We do not recommend storing the cells at -80 degrees C. Once thawed and transferred into room temperature medium in a vented, non-baffled shake flask, we recommend incubating the cells in a non-humidified, non-CO2 atmosphere incubator at 27 degrees C on a shaker platform set to 125±5 rpm for a shaker with a 19 mm or 25 mm orbital diameter or 95±5 rpm for a shaker with a 50 mm orbital diameter. Cells should exhibit ≥80% viability at 3 days post-thaw and should reach their normal 24 hour doubling time within 1-2 passages post-thaw.
ExpiSf9 cells should exhibit ≥80% viability at 3 days post-thaw. Within 1-2 passages post-thaw, the cells should be growing with a doubling time of approximately 24 hours. When cells are cultured at 0.5 x 10E6 cells/mL, or 1.0 x 10E6 cells/mL, viable cell density should be approximately 5-10 x 10E6 cells/mL within 3 or 4 days, respectively. If cells are not growing within these approximate ranges within 3 passages from thawing, cell culture conditions will require further optimization.
ExpiSf9 Cells do not reach log phase growth until a cell density of approximately 4 x 10E6 cells/mL. Therefore, the cells should be allowed to attain a density of 5-10 x 10E6 cells/mL before subculturing to help ensure they have reached log phase growth. For all cell manipulations, we recommend simply swirling flasks to resuspend the cells, not shaking or pipetting the cells vigorously to mix, as this can lead to decreased performance, especially just prior to transfection when cells have attained very high densities.
ExpiSf9 Cells should be passaged at least three times post-thaw and be growing within the ranges specified in the product manual, prior to transfection and/or viral infection. The cells should maintain consistent performance for at least 20 passages if maintained in accordance with the cell culture maintenance guidelines in the product manual. For optimal performance, we recommend thawing a new vial of cells after 30 passages.
As a quick check, we recommend seeding ExpiSf9 Cells at 0.5 x 10E6 cells/mL in 25 mL of ExpiSf CD Medium in a 125 mL vented, non-baffled shake flask and checking viability and viable cell density on Days 5, 6, and 7 post-seeding. Typically, ExpiSf9 Cells will reach maximal cell density, in the range of 18-20 x 10E6 cells/mL, around Day 7 post-seeding. The cells will continue to show high viability from Day 7 to Day 8, after which there will be a drop in viability.
Note: The final viable cell density value 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 shake speeds simultaneously to determine which speed provides optimal cell growth and then start with this speed for protein expression runs.
The ExpiSf Expression System allows rapid generation of high-titer baculovirus using a suspension-based transfection protocol. ExpiSf9 Cells should be seeded at a density of 2.5 x 10E6 viable cells/mL just prior to transfection. After seeding, cells are returned to the incubator for 0-30 mins and then transfected following the recommended protocol. If desired, ExpiSf9 Cells can also be transfected in adherent culture format. When using adherent ExpiSf9 cell culture, cells should be seeded at a density of 1 x 10E6 cells/well in 6-well plates just prior to transfection. After seeding, cells are returned to the incubator for 30-60 minutes to allow them to adhere to the plate, and then transfected following the recommended protocol.
We suggest using a baculovirus stock with a titer of >1 x 10E8 infectious virus particles (ivp)/mL for protein expression studies. Using the recommended suspension-based transfection protocol, the typical range of P0 virus titer that we have typically observed is 1-5 x 10E9 ivp/mL (4 days post-transfection).
We don't recommend amplifying the baculovirus stock higher than P3, as more defective interfering particles will be produced and a decrease in protein expression level will occur. Notably, we have observed a gradual decrease in baculovirus infectivity at each amplification step; therefore, we recommend avoiding multiple rounds of amplification. Instead, we recommend following our suspension-based transfection protocol using the ExpiFectamine Sf Transfection Reagent so that you can generate large quantities of high-titer, high-quality P0 virus.
We recommend using a FACS-based method for quantifying gp64-positive, infected cells, as described in the ExpiSf Expression System User Guide. This method allows you to determine the titer of your recombinant baculovirus stock in infectious virus particles (ivp) per mL of culture in less than 24 hours.
One day prior to virus infection, ExpiSf9 Cells should be seeded at a density of 5 x 10E6 viable cells/mL and immediately treated with ExpiSf Enhancer (Day1). On the next day (Day 0; 18-24 hrs after enhancer addition), the cells should be at a density of 5-7 x 10E6 cells/mL and ready for infection. It is important to infect cells within the 18-24 hr period after enhancer addition, for optimal performance.
Note: Incubating ExpiSf Enhancer-treated ExpiSf9 Cells for longer than 24 hrs may result in decreased infection efficiency and low protein yields.
For the best results, we recommend adding ExpiSf Enhancer at the time of cell seeding, 18-24 hrs prior to virus infection. The enhancer may be added to the flasks without pre-warming.
When ExpiSf9 Cells are efficiently infected, viable cell density will decrease to <80% at Day 3 post-infection. In parallel, cell diameter will increase from 16 µm (uninfected) to 19-20 µm at Day 3 post-infection. Finally, when observed under a microscope, infected cells will have enlarged nuclei and the cytoplasm may contain vacuoles and demonstrate granularity.
We recommend using an MOI of 5 for expression studies using ExpiSf Cells. However, when expressing a protein for the first time, you may want to follow the recommendations below to optimize titer of your recombinant protein:
- MOI: Infect cells at varying MOIs (e.g., 1, 2, 5, and 10) and assay for protein expression.
- Baculovirus volume: Infect cells using varying volumes of baculovirus (e.g., 100 µL, 200 µL, 500 µL, 1 mL) and assay for protein expression.
- Time course: Infect cells at a constant MOI or volume, and assay for recombinant protein expression at different time points post-infection (e.g., 24, 48, 72, 96 hours post-infection).
Yes. We offer the ExpiSf Expression System which is highly scalable with a range of protocols available, spanning from 24-deep-well plates to at least 3 L shake flasks. In addition, larger scale growth of ExpiSf9 Cells in ExpiSf CD Medium is possible.
The time of harvest is highly dependent upon the nature of the protein. Because baculovirus is a lytic virus, infected cells will eventually lyse. It is important to determine the expression kinetics for each protein, as many proteins (secreted or non-secreted) may be degraded by cellular proteases released in cell culture. Maximum expression is usually observed between 30 and 72 hrs post-infection for secreted proteins and between 48 and 96 hrs post-infection for non-secreted proteins. For some proteins, you may need to protect the recombinant protein from proteolysis by supplementing cultures post-infection and/or at the time of harvest with protease inhibitors. We recommend using Halt Protease Inhibitor Cocktail (Cat. No. 87786).
Yes, you may be able to adapt your Sf9 cells for growth in ExpiSf CD Medium. We recommend following a serial adaptation protocol to slowly acclimate your cells for growth in ExpiSf CD Medium. A detailed cell adaptation protocol is provided in the ExpiSf CD Medium User Guide. Long-term adaptation in ExpiSf CD Medium may increase productivity of your Sf9 cells, and should support high-density growth. However, there is no guarantee that other insect cell lines will achieve the same levels of baculovirus and protein expression as the ExpiSf9 Cells. In limited testing, we have found other Sf9 and Sf21 cell lines to achieve lower maximum cell densities when fully adapted to ExpiSf CD Medium with lower protein yields. The performance of your own insect cell line in ExpiSf CD Medium will need to be empirically determined.
All three media formulations are protein-free and serum-free and optimized for Sf9 and Sf21 cell culture and protein expression. However, ExpiSf CD Medium is a chemically defined, yeastolate-free formulation whereas both Sf-900 II SFM and Sf-900 III SFM media contain hydrolysates, therefore making them undefined formulations. The absence of hydrolysates or any other undefined component makes ExpiSf CD Medium a superior formulation with greater lot-to-lot cell growth and protein expression consistency run after run. In terms of maximum cell densities, ExpiSf CD Medium can support the highest maximum Sf9 cell density of approximately 20 x 10E6 cells/mL whereas Sf-900 II SFM and Sf-900 III SFM support maximum Sf9 cell densities of 10 x 10E6 cells/mL and 14 x 10E6 cells/mL, respectively.
ExpiSf CD Medium is a core component of the ExpiSf Expression System and has been designed to support high-density growth of suspension ExpiSf9 cells. The medium supports superior baculovirus generation and protein expression as part of the ExpiSf System. You may adapt your own Sf9 cell line for growth in ExpiSf CD Medium using a sequential adaptation protocol described in the ExpiSf CD Medium User Guide. However, there is no guarantee that other insect cell lines will achieve the same levels of baculovirus and protein expression as the ExpiSf9 Cells.
Both adherent- and suspension-based transfection protocols will allow you to generate high-titer baculovirus stocks using the ExpiSf Expression System. You may use either protocol interchangeably depending on your lab capabilities and preference. The use of suspension culture versus adherent culture can increase the cell density per mL of culture, and therefore will allow you to generate a larger amount and volume of P1 virus stock in shorter period of time.
No. The ExpiFectamine Sf Transfection Reagent is a low-toxicity reagent and is designed to be used without media exchanges. There is no need to remove transfection complexes or to change growth medium following transfection.
ExpiFectamine Sf Transfection Reagent typically exhibits lower toxicity and a faster complexation time and has a simpler protocol compared to other DNA transfection reagents. Lower toxicity means that no media change is required post-transfection. Shorter complexation time and simpler protocol means that DNA:lipid complexes are formed in only 5 mins and undiluted DNA is added directly to diluted ExpiFectamine Sf solution. This translates to time savings and the streamlining of your transfection reactions. This can be particularly advantageous where a higher throughput is required.
For Research Use Only. Not for use in diagnostic procedures.