Baculovirus Expression Services
Starting from a transfer vector (we work with a number of different transfer vectors - please inquire), two independent recombinant baculovirus P0 stocks (approximately 2 ml) will be generated. Each P0 stock will be amplified in Sf9 cells to yield 2 x 100 mL P1 stocks. 1 mL of each P1 stock will be used to infect 50 mL of Sf9 cells and 25 mL culture samples will be taken at 48- and 72-hours post infection. For each time point, cells will be harvested, flash frozen in liquid nitrogen, and stored at -80°C. For secreted proteins, we will harvest media and store at 4 C.
Delivered: 2x P0 (1.5 mL), 2x P1 stock (100 mL), P1 expression test cells pellets (or media) 2x 48 hpi + 2x 72 hpi.
Time: 14-21 days.
Baculovirus Expression Testing
(A) His-tagged or GST-fusion proteins: Lysis in buffer (standard lysis buffer is 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 10% glycerol; 0.5 mM TCEP; protease inhibitor cocktail), clarification by centrifugation, mixing with either Ni-NTA- or glutathione-agarose, recovery and washing of affinity resin. Elution (imidazole or reduced glutathione, respectively) and analysis by SDS-PAGE.
(B) Western Blot: Lysis in standard buffer (above), clarification by centrifugation followed by fractionation of clarified lysate by SDS-PAGE and blotting to nitrocellulose membrane. Blot will be blocked and treated with primary antibody supplied by customer (or anti-FLAG Ab) with washing (Tris-buffered saline with Triton X-100) and treatment with secondary antibody (goat anti-mouse peroxidase conjugate) and staining using DAB (diaminobenzadine) as substrate.
Delivered: Summary of expression experiment including annotated images of stained SDS-gel or developed western blot.
Time: 2 days.
Baculovirus Titer Determination
We perform a limiting dilution virus titer assay using Sf9 cells in a 96-well format. This assay is the most powerful method to determine virus titer because it scores for virus. Other fast methods score for secondary markers that can be inaccurate. Baculovirus stock will be diluted into a 10-fold series and used to infect Sf9 cells in a 96-well plate format. The number of infected wells will be scored at 6-7 days post infection and TCID50 converted to pfu/mL.
Delivered: Virus titer determination in pfu/ml.
Time: 1 week.
A 1/500th volume of P1 or P2 viral stock will be used to infect Sf9 cells (at density = 1e6 cells/mL). Following 4-5 days post infection (viability < 50%), the culture media will be clarified by centrifugation and stored at 4 C. The resulting virus stock is sterile filtered to remove cell debris that can inactivate infectious virions.
Delivered: Amplified baculovirus stock in sterile container.
Time: 1 week.
Baculovirus-Infected Insect Cell Fermentation
Sf9 or T.ni cells cultures will be expanded to target volume and density (1-25 L; 1-2e6 cells/mL) and infected with 1/100 volume of amplified virus stock. Cultures are harvested at peak protein expression time post infection (as determined by expression test or customer information). Cell pastes will be flash frozen in liquid nitrogen and stored at -80 C.
Delivered: Frozen cell pastes and summary of fermentation data.
Time: 1 week for 1-5L | 2 weeks for 10-25 L.