Insect cells and lytic baculoviruses provide a proven method for high-level expression of full-length mammalian proteins. Autographa californica nuclear polyhedrosis virus (AcNPV) is used to infect cultured insect cells (eg, Spodoptera frugiperda). Expression of the very abundant polyhedrin gene is not essential in tissue culture and its strong promoter can be used for the transcription of foreign genes.
The polyhedrin promoter is maximally expressed at a very late stage in infection when the lytic virus kills host cells, resulting in high levels of expression even for certain toxic proteins. Also, many post-translational modifications similar to those of mammalian cells are made in Insect cells and proteins that cannot be expressed in E. coli have it has been successfully expressed in the insect cell system.
Sapphire ™ baculovirus DNA offers the following benefits:
- Improved protein folding. The disulfide isomerase gene was inserted into the p10 locus of the virus to ensure adequate protein formation of disulfide bonds.
- High recombination efficiency. Baculoviral Sapphire ™ DNA contains a lethal deletion of ORF1629 that can only give rise to viable viral particles if rescued by homologous recombination with a transfer based on a polyhedrin promoter vector. This design significantly reduces time and effort in plaque assays.
- High-level expression. The p10 promoter is partially inactivated and the lytic gene p10 is removed so that transcription levels are higher due to reduced interference and healthier insect cells.
Co-transfection with the Sapphire ™ Transfection Kit
1. Sow 2 x 106 Sf9 (Cat. No. CEL-10001) or Sf21 (Cat. No. CEL10002) cells in each 60 mm tissue culture plate. Allow cells to attach firmly, which usually takes 5-10 minutes.
2. Remove the medium and replace it with 1 ml of sterile transfection. Sapphire ™ Insect Transfection Kit Buffer A (Cat BVD-10003).
3. Mix 0.5 µg of Sapphire ™ DNA and 2 µg of Recombinant baculovirus transfer vector containing the gene of interest in a microcentrifuge tube.
4. Allow the mixture from Step 3 to stand for 1 minute at room temperature before mixing adding 1 ml of sterile Sapphire ™ Transfection Buffer B Insect Transfection Kit (Catalog # BVD-10003).
5. Mix well and add the mixture from Step 4 dropwise to the cells of Step 2. For the negative control, use Sapphire ™ DNA alone without any DNA vector. For positive control, use pVL1392-XylE (Cat. No. BVP-10001) in combination with Sapphire ™ DNA.
6. Incubate plates at 27 ° C for 4 hours. After that time, delete medium and replace with fresh TNM-FH medium containing 10% Fetal Bovine Serum (Cat. No. MED-10001).
7. After 5 days, collect the supernatant from all three plates and infect fresh cells to amplify the virus. Optionally, lyse the transfected cells and check the expression of your protein of interest.
Recombinant baculovirus amplification
After cotransfection, the recombinant baculovirus must be amplified to obtain a high titer stock solution. To achieve this. Newly seeded insect cells must be infected in a multiplicity of infection (MOI) of <1. This is usually done by infecting 5 x 106 cells per 10 cm plate (approximately 60% confluence) with 500 µl of transfection supernatant in 15 ml of TNM-FH medium supplemented with 10% FBS (Cat. No. MED-10001).
It must be incubated at 27ºC for 3 days before harvesting. At 24h after infection, virus-infected cells are visibly swollen with enlargement nucleus that can be observed by light microscopy. The medium will contain at least 107 virus particles per ml and must be used for another round of amplification. Two rounds of amplification typically give a virus titer of 2 x 10 8 virus particles per ml of medium.
Recombinant Baculovirus Storage:
Use the following procedure for long-term storage of virus stocks:
- Centrifuge the viral stock at 4000 x g to remove cellular debris.
- If the medium does not contain serum, add 10% serum.
- Store viral stocks at + 4 ° C. However, the virus titer may decrease 5- 10 times during 6 months of conservation at 4ºC.
- Protect viral stocks from light to ensure maintenance of the title.
- For long-term storage (up to two years), store small aliquots of viruses to -80 ° C. Avoid repeated freezing and thawing