I cannot detect any recombinant fusion protein after using the BaculoDirect Expression Kit. What could be the cause for this and what do you suggest I try?
Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.
I'm getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?
To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.
I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do?
Please see our recommendations below:
- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.
I see a precipitate in my ganciclovir solution. What can I do?
Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.
I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?
Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.
Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.
My viral stock used to work well from my Bac-to-Bac baculovirus expression system, but after a couple of months, there is much less protein expression. Why is this?
Please review the following possibilities and solutions:
- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.
I'm getting low protein yield when using the Bac-to-Bac baculovirus expression system. What are the possible reasons for this?
Low protein yield may occur due to the following reasons:
- Viral stock contains a mixture of recombinant and non-recombinant baculovirus: Perform plaque purification to isolate recombinant baculovirus.
- Baculovirus is not recombinant: Verify transposition by PCR analysis of bacmid DNA using pUC/M13 forward and reverse primers; re-transfect insect cells with new recombinant bacmid DNA.
- Use too low or too high viral titer: Vary the MOI.
- Time of cell harvest is not optimal: perform a time course of expression to determine the optimal time to obtain maximal protein expression.
- Cell growth conditions and medium are not optimal: Optimize cell culture conditions based on the size of your culture vessel and expression conditions; we recommend using Sf-900 II SFM or Sf-900 III SFM for optimal cell growth and protein expression.
- Cell line is not optimal; try other insect cell lines.
- Cells were harvested too late: Do a time-course experiment and harvest cells at different time points.
I don't see any signs of cell infection 72 hours after infection with my P2 viral stock. Why is this?
Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.
Using the Bac-to-Bac baculovirus expression system, I've transduced my cells but do not see any signs of viral production. It has been 5 days. What happened and what should I do?
Please see the possible reasons and suggestions below:
- Mixture of Cellfectin II Reagent and bacmid was not performed or was not incubated long enough: Mix the Cellfectin II Reagent and bacmid well by tapping or gentle vortexing, and incubate the mixture for 15-45 min.
- Bacmid yield is lower than estimated: Set up an optimization with different amounts of bacmid.
- Bacmid is sheared during purification or freeze/thaw: Verify the integrity of bacmid on a gel.
- Incubation time is not long enough: Incubate mix for 8 hr at 27 degrees C.
- Cells used are of high passages or have passed log-phase growth: For best results, use cells between 8-15 passages; plate cells when they are in log-phase growth.
- Cellfectin II Reagent has been frozen: Purchase a new vial.
- Medium used contains serum: Use unsupplemented Grace's medium in transfection.
Both transfected cells and Cellfectin II Reagent alone negative control cells are dead within 36 hours. What could cause this?
There are several possibilities:
- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.
My transfected cells are floating up and look dead, but the Cellfectin II Reagent alone control plate is fine. What could be happening?
This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin II Reagent. Additionally, use the PureLink HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.
My bacmid DNA contains a mixture of recombinant bacmid and empty bacmid. What am I doing wrong?
Most likely, a colony that was gray or dark in the center was picked. Try to analyze more white DH10Bac transformants. Typically, we recommend picking a white colony whose diameter is >2 mm. Restreak the white colonies on a fresh plate with 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal and 40 µg/mL IPTG. Incubate plates for 24 hours.
My PCR analysis of the recombinant bacmid gives no product. What could be the cause of this and do you have any recommendations on how to fix this?
Please see the possible causes and suggestions we have to alleviate this problem:
- Insert is very long and causes difficulties in PCR: Instead of using both M13 forward and reverse primers, use one gene-specific primer paired with the M13 primer of your choice.
- Long GC-rich stretches in the gene of interest: Consider using DMSO (up to 8%) in the PCR reaction.
My bacmid DNA is degraded. What could be the cause for this and what recommendations do you have to fix this?
Please see the possible reasons and suggestions below:
- DNA stored improperly: Ensure that purified bacmid DNA is stored at -20 degrees C in aliquots to avoid repeated free/thaws.
- High molecular weight bacmid DNA handled improperly: When isolating bacmid DNA, do not vortex the DNA solution; additionally, do not resuspend DNA pellets mechanically; allow solution to sit in the tube with occasional tapping.
I am trying to isolate my bacmid DNA but the overnight culture did not grow. What could have happened?
This could be caused by the following:
- Wrong antibiotic or old media: use fresh media.
- Colonies are too old or too small: Use large white colonies from freshly streaked plates.
- Unstable insert caused by special feature of the gene of interest; for example, direct repeats: Incubate the culture at 30 degrees C for 24 hours instead of 37 degrees C overnight.
I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and am seeing very poor blue/white colony differentiation. What should I do?
Poor color differentiation for your colonies could be caused by the following:
- Agar is not at the correct pH: Adjust pH of LB agar to 7.0.
- Intensity of the blue color is too weak; ensure that you are using Bluo-gal, not X-Gal. You can also try increasing the concentration of Bluo-gal to 300 µg/mL.
- Too many or too few colonies on the plate: Adjust the serial dilutions of cells to obtain an optimal number of colonies.
- Incubation period too short or temperature too low: Do not pick colonies until 48 hours after plating; incubate plates at 37 degrees C.
- IPTG concentration is not optimal: A range of 20-60 µg/mL IPTG generally gives optimal color development.
I transfected my bacmid DNA but saw no signs of infection. The quality of my DNA prep might be an issue. How can I analyze the bacmid DNA?
We recommend running 1/8th of the 40 µL midiprep sample on a 0.5% TAE agarose gel. Electrophorese slowly at 23 V for 12 hr. The banding pattern of the recombinant bacmid midiprep should be seen.
I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and I'm getting very few colonies. What could be the cause for this and do you have any recommendations for how to fix this?
Please review the following reasons and our recommendations:
- Use LB medium for recovery/expression period: Use SOC medium for the 4 hr growth time.
- Recovery/expression time too short: Increase the recovery time to > 4 hr at 37 degrees C or 6 hr at 30 degrees C.
- IPTG concentration is not optimal: We suggest using 20-40 µg/mL IPTG.
I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and all of my colonies are blue. What could be the cause of this and what should I do?
Please review the following possibilities and recommendations:
- pFastBac DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac expression system and all the colonies obtained are white. Shouldn't I expect to see some blue colonies?
Although you will be picking white (recombinant) colonies, you should expect to see some blue (contain non-recombinant bacmid) colonies. Here are some possible causes for seeing no blue colonies and recommendations for the same:
- Insufficient time for color development: Wait at least 48 hours before identifying colony phenotypes.
- Use Bluo-gal instead of X-Gal in agar plates: Use Bluo-gal in plates to increase contrast between blue and white colonies.
- Insufficient growth after transposition: Grow transformed cells in SOC medium for a minimum of 4 hours before plating.
- Bluo-gal and IPTG omitted from plates: Prepare fresh selective plates containing 50 µg/mL kanamycin, 7 µg/mL gentamicin, 10 µg/mL tetracycline, 100 µg/mL Bluo-gal, and 40 µg/mL IPTG.
- There are too many colonies on the plate: Serially dilute the transformation mix to obtain well-spaced colonies (10-2 to 10-4 is suggested).
- Plates are too old or stored in light: Do not use plates that are more than 4 weeks old; store plates protected from light.
- Incubation period too short or temperature is too low: Wait at least 48 hours before picking colonies. Incubate plates at 37 degrees C.
What is the difference between the Bac-N-Blue expression system and the Bac-to-Bac 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.
What steps are recommended for preventing proteolysis in the baculovirus expression systems?
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.
Is there a packaging limit for the baculovirus?
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.
Are there preferred secretion signals for use with baculovirus?
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.
I'm interested in expressing a large protein, larger than 130 kDa. Can I use the baculovirus system or will the proteins be degraded?
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.
What is the size of the baculovirus polyhedron protein?
The polyhedron protein is 30 kDa.
What is the promoter used in the Bac-to-Bac expression system?
The promoter that drives the gene of interest is the polyhedron promoter.
Which strand is produced using M13K07 to make ssDNA from pPROEX HT or pFastBAC vectors?
You will get the antisense strand with pProEXHT.
You will get the + or sense strand with pFastBac I of pFastBac HT.
