Protein Expression in Insect Cells

2.1.1.\ Insect Cell Culture and Infection with Baculovinis

The insect cells used in this study were sf9, sf21, Hi5 cells {Spodoptera frugiperda cells), which were derived from pupal ovarian tissue o f fall armyworm. Sf9

and sfZl cells were cultured in the 500 ml spinning bottle at 27 °C with constant stirring at 120 rpm in suspension, in TC-lOO medium containing 10% fetal calf serum (PCS), 100 U/500 ml penicillin and 100 U/500 ml streptomycin (the resulting medium was called complete TC-lOO medium), up to a density o f 1x10® cells/ml. Cells were then seeded at the required density onto petri dishes and allowed to adhere for 30 minutes at room temperature before infected with virus. Media was removed from cells by aspiration, prior to infection by adding virus stock drop-wise onto the cells and incubating at room temperature with gentle rocking for 1 hour. Afrer remove the viral solution from the infected cells by aspiration, plates (90 mm) were then supplemented with 4-10 ml more TC-lOO completed medium, and incubated at 27 °C with humidity for 4 days, unless otherwise stated. HiS cells were always cultured in monolayer in flasks using the same media under the same conditions, and were infected by the baculovinis using the same method. Alternatively, the cells were cultured in the serum free conditions by using SFM-II medium containing the same amount o f the antibiotics. The medium was changed every 3-4 days.

1.1.1,2 Cationic Liposome M ediated Transfection of Insect Cells

1 ml o f TC-lOO medium (containing no supplements) was incubated with 1 pg o f linear Autographa Californica nuclear polyhedrosis virus (AcMNPV) DNA, 3 pg o f plasmid DNA (apoCIII in transfer vector) and 20 pi o f the Cationic Hposomes solution (Invitrogen, San Diego, CA, USA) at room temperature for 15 minutes. This mixture was then added to 2x10^ o f log phase st9 cells in 60 mm plates and slowly rocked at room temperature for 4 hours, following by adding 1 ml o f TC-lOO complete medium into the plates and then incubated at 27 °C in a humidified

incubator. The medium in the plates was harvested in 48 hours and stored at 4 °C as virus stock.

2.2,13 Plaque Purification o f Recombinant Virus and the Determination of the

Virus Titer

1.5x10® o f log phase s© cells in 35-mm plates were infected with 100 pi o f the virus stock by incubating at room temperature for 1 hour. The virus stock was then removed con^letely by aspiration, followed by adding 1.5 ml o f 1% Sea Plaque Agarose solution (pre-made and incubated at 37 °C, by mixing 2% aqueous Sea Plaque Agarose solution with equal volume o f Grace medium containing 150 pg/ml o f 5-bromo-4-chloro-3-indolyl-p-D-galactoside (X-gal)) into the dishes. These dishes were then left at room temperature for approximately 30 minutes to allow the agarose to set before being incubated in a humidified incubator at 27 °C. Blue plaques appeared after 5-6 days incubation.

For the pure baculovirus, this method can also be used detect the viral titer (pfii/ml) according to the number o f the plaque obtained.

2.2.7.4 Amplification of the Virus from the Plaque

Viral occlusion bodies are formed in the nucleus and comprise enveloped nucleocapsid embedded in a crystalline protein matrix. They have a bright shiny appearance that is readily visualised under the light microscope. Blue plaques without occlusion bodies in the insect cells (occ-) were picked using a sterile Pasteur pipette and bulb. The agarose plugs were soaked in 1 ml o f complete TC-lOO medium overnight and then votexed. The resulting mixture was stored at 4 °C as plaque-pick. In order to amplify the virus in the plaque-pick, 100 pi o f the plaque-pick solution was used to infect 5x10^ o f log phase s© cells at room temperature for 1 hour. 2 ml o f the complete TC-lOO medium was then added into the plates in prior to incubate at 27 °C in a humidified incubator. The medium covering the cells was harvested after 4 days

and stored as Pass-1 virus stock after centrifiigation at 1500 rpm to pellet the cell debris. Pass-2 virus stock was made in the same way except that 5x10^ o f log phase sf9 cells were inoculated with the 0.5 ml o f the Pass-1 stock.

The amplification o f the large scale o f the pure virus was performed as same as described above except using pure virus instead o f the plaque-pick solution.

2.2.7.5 Extraction of Viral DNA

0.75 ml o f virus stock (normally Pass-2) was mixed with an equal volume o f 20% PEG solution in 1 M NaCl in a fresh 1.5 ml eppendorf tube by inverting twice and the resulting mixture was allowed to stand at room temperature for 30 minutes. After the mixture was spun at 14000 rpm for 10 minutes at room temperature, all the supernatant was removed. The remaining pellets were then dissolved in 100 pi o f sterile H2O followed by addition o f 10 pi o f Proteinase K (5-10 mg/ml). The resulting

solution was then incubated at 50 °C. After 1 hour, the solution was extracted with an equal volume o f phenol/chloroform (1:1) to remove aU the proteins. 10 pi o f 3M NaOAc (pH5.2), 5 pi o f glycogen and 220 pi o f ethanol were added and the resulting mixture was incubated at -20 °C. After at least 20 minutes, the above mixture was spun at 14000 rpm for 15 minutes at 4 °C and all the supernatant was removed. The remaining pellets were washed with 0.5 ml o f 80% ethanol and Spun at 14000 rpm for 5 minutes to remove all trace o f ethanol. The final viral DNA pellets were either stored at 4 °C for a day or at -20 °C for longer periods.

2.2.7.Ô Time Course of Recom binant A poCIII Expression

5x10^ o f log phase insect cells were grown in the 6-well plates and infected

with pure baculovirus at multiplicity o f infection (MOI) (plaque forming units/cell) o f 5, 7.5 and 10 for 1 hour. Amount o f titered virus (detected as described in section 2.2.13) needed to infect the cells was calculated as the equation:

ml o f virus = MOI x 5x10^ (number of cells) titer (pfii/ml)

Cells were then washed 3 times with TC-lOO medium (no serum) to remove all FCS contained in the TC-lOO complete medium used previously and finally covered with 3 ml o f SFM-II serum fi’ee medium. At 12-hour time points Jfrom 0-96 hours after infection, 500 pi o f medium were removed and centrifuged at 1500 rpm for 5 minutes. Additionally, the cells scratched fi*om one well were lysed by sonication in 200 pi o f PBS containing 1 mM o f PMSF for 3x15 seconds bursts at a medium intensity setting while holding the suspension at ice bath. Above sanqjles o f both the medium and the cell lysate were stored at -20 °C until all the samples o f each time point were obtained and then used for the detection o f the expression level. Finally, 25 pi o f the medium and 5 pi o f the cell lysate on 17.5 % precast Tricine-SDS-PAGE as described in section 2 .2.6.1.