The overall goal of this protocol is to produce baculovirus in insect cell culture, purify with heparin affinity chromatography and to concentrate with ultracentrifugation. The final product can be used for in vivo gene transfer or as a vaccine. This method can help researchers who want to use purified and concentrated baculovirus for gene therapy application.
Baculovirus particles may contain cellular debris and proteins from Sf9 culture which may induce inflammation or an immune response when used in people. To avoid toxicity, baculovirus is purified and separated from contaminating particles. Some parts of the procedure will be demonstrated by Marilou Narciso, a research assistant from my laboratory.
To set up the suspension culture, seed 50 milliliters of Sf9 cells in insect culture medium into a 250 milliliters polycarbonate Erlenmeyer flask. And place the flask in an orbital shaker incubator. Add two milliliters of P2 baculovirus stock to the cells, and shake at 135rpm while maintaining a constant temperature of 28 degrees Celsius.
Four days after infection, the entire Sf9 cell population will show cytopathic effect, which is an indication of high titer baculovirus production. Sequentially amplify the baculovirus until the desired volume is obtained, by increasing the volume of cell culture in each subsequent infection 10 to 50-fold. After centrifuging the final baculovirus supernatant for 30 minutes at 1000g and four degrees Celsius to separate the course cellular debris, transfer the supernatant to a clean vessel and treat with nuclease at 50 units per milliliter for two hours at 37 degrees Celsius to digest the genomic DNA and RNA.
Finally, filter the viral supernatant through a 0.45 micron filtration unit. Set up the chromatography system by placing the wash buffer line from inlet port A1 into a bottle containing at least 500 milliliters of wash buffer. Use the A2 buffer inlet port for loading the elution buffer.
Insert several 50 milliliters conical tubes into the fraction collector to collect the column pass-through baculovirus supernatant, the wash buffer, and the eluded baculovirus. Equilibrate the heparin column with five 7.9 milliliters column volumes of wash buffer at a linear flow rate of seven milliliters per minute. Load 250 milliliters of baculovirus supernatant, onto the heparin column using the sample pump of the inlet sample port at a linear flow rate of two milliliters per minute.
Then run 10 column volumes of wash buffer through the heparin column at a linear flow rate of two milliliters per minute until the ultraviolet absorbance curve has returned to baseline and become stable. Elude the baculovirus particles from the heparin column with five column volumes of elution buffer at a linear flow rate of four milliliters per minute. Watch for a sharp elution peak of protein on the chromatogram when the baculovirus particles dissociate from the heparin column.
Post elution, immediately dilute the eluded baculovirus supernatant 10-fold using 20 millimeter sodium phosphate buffer to prevent an activation of baculovirus particles from osmotic shock during subsequent centrifugation. Begin the concentration procedure by adding an equal volume of the baculovirus supernatant to autoclave to ultracentrifuge tubes. Measure the tube weight by a balance and adjust the weight of the contents of the ultracentrifuge tubes with sterile PBS.
Place each of the ultracentrifuge tubes in an opposing bucket of the SW 32 Ti rotor and run the ultracentrifuge at 80, 000g for 90 minutes at four degrees Celsius. Following centrifugation, open the ultracentrifuge tubes in a tissue culture hood and aseptically aspirate the liquid without removing the baculovirus pellet. Add 200 microliters of PBS containing 0.5%BSA and resuspend each pellet by vigorously pipetting up and down.
Finally, transfer 50 to 100 microliter aliquots of the concentrated baculovirus to cryovials and store at negative 80 degrees Celsius. Baculovirus titers were estimated by infection of HT1080 cells. The line connected by diamonds shows the total infectious units of baculovirus in each fraction.
Baculovirus particles strongly bind to the heparin column, therefore no significant amount of virus is detected in the column wash buffer. A sharp peak of protein is observed during elution, which corresponds to the baculovirus fraction. The parameters used in this protocol maximize the yield and minimize the inactivation of infectious baculovirus particles.
This protocol can be used for purification of other viruses and proteins that have an affinity for the heparin.
