Recombinant proteins have been produced in the Baculovirus system at levels rang- ing between 0.1% and 50% of the total insect cell protein. For optimal protein pro- duction, the MOI should be between 3 and 10. Researchers should test different MOI to empirically determine optimum levels for protein production. The supernatant from protein production should not be used as a viral stock. Since the MOI used was much higher than one, a considerable portion of the virus population may contain deletion mutations. Several variables influence protein levels, functional activity and post-trans- lational modifications of Baculovirus-expressed protein (refer to Chapter 4.2). An exam- ple of the variation of expression levels between four proteins–cyclin A, cdk2, TR2 orphan receptor, and androgen receptor–is seen in Fig. 10(A). The percent of protein expressed in the system is highly dependent on the intrinsic property of the protein. The cyclin A from Fig. 10(A) was not visible by Coomassie blue-staining, and was analyzed by western blot analysis Fig. 10(B). Figure 10(C) shows post-translationally modified IL–4. A comparative analysis of phosphorylated Baculovirus-expressed and native retinoblastoma protein (Rb) is shown in Fig. 11. Figure 12 shows assays used to mea- sure the functional activity of Baculovirus-expressed granulocyte macrophage colony stimulated factors (GM-CSF) and Interleukin-4 (IL-4). We suggest that the user consult the literature pertinent to their recombinant protein to gain information regarding expected post-translational modifications and levels of functional activity.
Figure 10. Examples of recombinant protein expression levels in Baculovirus-infected Sf9 cells.
A) Protein expression levels. Amido black SDS-PAGE of total insect cell lysate (20 µg/lane) containing Bac- ulovirus-expressed cyclin A (lane 1), Cdk2 (lane 2), TR2 (lane 3), or androgen receptor (lane 4). B) Western blot analysis of Baculovirus-expressed cyclin A. Anti-cyclin A monoclonal antibody (clone BF683, Cat. No. 14531A) (lane 1). Isotype (negative) IgE control (lane 2). C) SDS-PAGE analysis of Baculovirus-expressed, purified mouse IL-4. IL-4 was purified using an anti-mouse IL-4 monoclonal antibody (clone BVD6-24G2, Cat. No. 18041D). The gel was stained with coomassie blue. Note that although Baculovirus-expressed cyclin A was not visible by staining (A, lane 1) it was readily visible by western blot analysis (B, lane 1). IL-4 migrates as two bands due to differential glycosylation (C).
For large-scale protein production, we have found that cell propagation in spinner flasks and protein production on tissue culture plates is optimal. Protein may be pro- duced in suspension, but often the levels are lower than on plates.
Monolayer Cultures
1. Seed several individual 15 cm tissue culture plates with 2.0× 107Sf9 cells per plate. Add fresh TNM-FH medium to make up a total of 30 ml media per plate. 2. Calculate the amount of virus needed using the formula: ml of inoculum
needed = MOI (pfu/cell) ×number of cells/titer of virus per ml.
3. Infect seeded cells with high titer recombinant Baculoviruses stock solution (virus titer should be 1-2× 108pfu/ml). For optimal protein production, the MOI should be between 3 and 10. Often researchers will test different MOIs to empirically determine the optimum level of infection.
15 cm tissue culture plate (Falcon Cat. No.3025) High titer viral stock (1.2×106 pfu/ml) 2.0× 107 Sf9 cells (Cat. No. 21300L) per plate
TNM-FH media (Cat. No. 21227M)
Materials Needed
✓
27°C Incubator kD 116 66 36 3 4Cyclin ACdk2 TR2 Androgen R
A 1 2 B IL-4 116 66 36 21 14 6 C
Anti-Cyclin AControl IL-4
1 2
SDS-PAGE Western Blot SDS-PAGE
116
66
36
4. Incubate the cells for 3 days at 27°C. Check for signs of infection 2–3 days after inoculation. Cells should be enlarged in size (about 2 fold) and a large nucleus should be visible.
5. Harvest the cells and supernatant from the plates and spin down the cells at 10,000× g for 5 min using a table-top centrifuge. Non-secreted proteins will be found in the cell pellet, which can be stored at –80°C. Secreted proteins will be found in the supernatant, which can be stored at –80°C. When purifying secreted protein, the cell pellet should be tested to determine the amount of protein, if any, that remains in the cells.
Suspension Cultures
1. Seed approximately 2× 106Sf9 cells/ml in a spinner flask. The cells should be healthy (98% viable).
2. Calculate the amount of virus needed using the formula: ml of inoculum needed = MOI (pfu/cell) x number of cells/titer of virus per ml. The desired MOI for pro- tein production is 3–10.
3. Add the inoculum to the flask. Incubate the flask at 27°C with stirring for 2-4 days. Check the progress of the infection by examining aliquots of the culture under the microscope.
4. To harvest, pellet cells by centrifugation. For secreted protein, store the super- natant in sterile tubes. For non-secreted proteins, store the cell pellet at –80°C and discard the supernatant.
TNM-FH media (Cat. No. 21227M) Spinner flask (Techne)
5 ×105 Sf9 cells per ml of culture (Cat. No. 21300L)
Hemocytometer (Fisher Cat. No. 0267110) Materials Needed
✓
27°C Incubator Spinner apparatus
Figure 11. Characterization of native and Baculovirus-expressed Retinoblastoma protein (Rb).
A) Western blot analysis of native Rb during different stages of the MOLT-4 (a human leukemia cell line) cell cycle. Native Rb migrates as multiple bands due to varying degrees of phosphorylation. Cell cycle stages are denoted as Q (quiescent), G1, S, and M. B) SDS-PAGE analysis of recombinant Rb. Rb is detected in Baculovirus-infected (lane 1) but not in mock-infected (lane 2) Sf9 cell lysates. The gel was stained with Coomassie blue. C) Comparative analysis of native and Baculovirus-expressed Rb by western blot. Rb expressed in MOLT-4 cells (lane 1) is more highly phosphorylated than Rb expressed in Baculovirus-infected Sf9 cells (lane 2). D) Analysis of phosphorylation in Baculovirus-expressed Rb. Baculovirus-infected Sf9 cells were labeled with 32P orthophosphate and treated or not treated with placental alkaline phosphatase (PAP). Both untreated (lanes 1a and 1b) and treated (lanes 2a and 2b) lysates were immunoprecipitated with anti-Rb antibody (clone G3-245, Cat. No. 14001A). Detection by autoradiography (top gel) shows that the radioactive label (lane 1a) is greatly reduced (lane 2a) following PAP-treatment. Western blot analy- sis of the autoradiographs (bottom gel) show that Rb in untreated lysates migrated at a higher molecular weight (lane 1b) than Rb in PAP-treated lysates (lane 2b). Collectively, the data indicate that Baculovirus- expressed Rb is phosphorylated, although at a lower level than native Rb. Abbreviations: pRb, under- phosphorylated Rb. ppRb, phosphorylated and highly phosphorylated Rb species.
1 2 110 ppRb pRb Rb 1 2 Sf9 Sf9 + Rb Rb A B C D 116 97 pRb + ppRb 1 2 3 4 Q G1 S M MOLT-4 MOL T-4 Sf + Rb – PA P + P AP Rb 1b 2b 1a 2a Sf9 + Rb kD
Figure 12. Functional activity of Baculovirus-expressed recombinant protein. hGM-CSF and
mIL-4 were cloned into pVL1393 and expressed in Sf9 cells. A) hGM-CSF assay. hGM-CSF activity was measured using the continuous cytokine dependent human cell line, TF-1.26hGM-CSF, at 10 µg/ml, was serially diluted 3 fold in 12 wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of TF-1 cells at 2× 105cells/ml were then added to each well for a final cell density of 2× 104/ml. After a 44 h incu- bation at 37°C in the presence of 5% CO2, the cultures were pulsed with 0.5 µCi tritiated thymidine (20 Ci/mM) for an additional 4 h. The cultures were then harvested and the incorporated thymidine mea- sured by scintillation counting. The data shown represent the cpm of thymidine incorporation versus 3 fold serial dilutions of hGM-CSF. Each point represents the mean of three replicates. B) Western blot analysis of hGM-CSF. Recombinant human GM-CSF (Cat. No. 19741V) loaded at 100 ng/lane and tested by Western blot analysis against purified anti-human GM-CSF (Cat. No. 18591D) at 1 µg/ml (lane 1) and normal rat serum at 1:500 dilution (lane 2). C) mIL-4 assay. IL-4 activity was measured using the contin- uous IL-2 dependent murine cell line, CTLL-2.27,28mIL-4 at 10 µg/ml was serially diluted 3 fold in 12 wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of CTLL-2 cells at 2× 105cells/ml were then added to each well for a final cell density of 2× 104/ml. After a 20-h incubation at 37°C in the pres- ence of 5% CO2, the cultures were pulsed with 0.5 µCi tritiated thymidine (20 Ci/mM) for an additional 4 h. The cultures were then harvested and the incorporated thymidine measured by scintillation count- ing. The data shown represents the cpm of thymidine incorporated versus 3 fold serial dilutions of mIL-4. Each point represents the mean of three replicates. D) Western blot analysis of mIL-4. Recombinant mIL- 4 lysate (Cat. No. 19231N) was loaded at 100 ng/lane and tested by Western blot analysis using purified anti-mouse IL-4 (Cat. No. 18031D) at 5 µg/ml (lane 1), and normal rat serum at 1:500 dilution (lane2).
A C D 55 200 97 116 66 36 31 21 14 1 2 55 200 97 116 66 36 31 21 14 1 2 B TF-1 Based GM-CSF Assay hGM-CSF CPM TdR Incorporated 0 10000 20000 30000 40000 50000 60000 70000 1 2 3 4 5 6 7 8 9 10 11 12
Serial 3-Fold Dilutions
CPM TdR Incorporated
CTLL-2 Based mIL-4 Assay mIL-4 0 2000 4000 6000 8000 10000 12000 1 2 3 4 5 6 7 8 9 10 11 12
Serial 3-Fold Dilutions
kD kD