Insect Expression Support—Getting Started

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.

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:

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 cell-only control. 
Granular appearance
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:

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

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 4^th day, collect P1 viral stock. Re-infect cells, and grow for 3 days. On the 7^th day, collect P2 viral stock. Infect cells, followed by harvesting of protein and purification on the 10^th 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.

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.

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 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.