Page 24 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 13

Protocol 1.8 Purification of recombinant virus and determination of viral titre by plaque assay

Materials 9. Transfer the plates from the incubator to the hood
Sf 9 cell culture and remove the medium.
100-mm and 12-well tissue culture plates 10. Add the insect cell medium at room temperature to
Baculovirus transfection supernatant the agarose solution and mix quickly.
11. Add 10 ml of the agarose solution prepared in Step 10
Agarplaque-Plus™agarose (BD Biosciences: Pharmingen)
to the side of each plate and cover the cell monolayer
EX-CEL 405™serum-free medium for insect cells containing
completely by gently tilting the plate.
50 µg/ml gentamycin sulphate and 2.5 µg/ml
12. Leave the plates undisturbed on a level surface until
amphotericin B
the agarose is set.
Sterile water containing 50 µg/ml gentamycin sulphate and 13. Place the plates in a sterile plastic box containing
2.5 µg/ml amphotericin B some sterile tissues sprayed with sterile water containing
A sterile plastic box that can accommodate six 100 ml tissue 50 µg/ml gentamycin sulphate and 2.5 µg/ml
culture plates amphotericin B.
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Microcentrifuge tubes 14. Incubate at 27 C for 6–10 days until visible plaques
Sterile pipette tips appear.
Microscope 15. Examine the plates for plaques using a microscope and
select a plate containing well separated plaques, and mark
SDS-polyacrylamide gel electrophoresis system
the position of each plaque with a marker pen.
Method 16. Count the number of plaques and calculate the number
1. Prepare 3% agarose solution in deionized water and of plaque forming units per ml virus stock.
sterilize by autoclaving. 17. Remove an agarose plug over the plaque using a sterile
2. Label six 100-cm plates each containing a monolayer pipette tip and place in a microcentrifuge tube containing
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of 2.1 × 10 cells. 1 ml insect medium.
3. Allow the cells to form a monolayer by placing the 18. Pick up 10–20 plaques as in Step 17 and place in
plate on a level surface. separate tubes.
4. Replace the old medium with 10 ml of fresh medium 19. Elute the virus from the agarose plug by rotating the
in each plate. tubes overnight in a cold room.
5. Prepare 10 −3 ,10 −4 ,10 −5 ,10 −6 , and 10 −7 dilutions 20. Add 200 µl virus from each tube to separate wells of
of the virus transfection supernatant. 12-well tissue culture plates each containing a monolayer
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6. Pipette 100 µl of the dilute virus transfection of 2 × 10 cells per well in 1 ml insect cell medium and
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supernatant onto each plate except for the control. incubate the plates at 27 C for 3 days.
7. Transfer the plates to 27 C incubator and leave 21. Collect the medium containing the virus from the wells,
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for 1 h. remove the cells by centrifugation at 1000 g for 5 min at
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8. Cool the agarose solution to 45 C and equilibrate 4 C, and store the virus at 4 C. Test the cells for protein
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two volumes of the medium to room temperature. expression by SDS-PAGE.
virus stock. At the end of the incubation the recombinant protein is where a large volume of the
recombinant protein level in the wells is compared cell culture is infected in a spinner flask and cells
by SDS-polyacrylamide gel electrophoresis. This are harvested by centrifugation (Protocol 1.11). The
method is quicker and easier than the plaque secreted protein is usually expressed using the insect
assay. cell medium that contains either a low concentration
For the large-scale expression of a non-secreted (2%) of fetal calf serum or serum-free insect cell
protein, cell monolayers in several tissue culture medium. The methods used for the purification of
flasks are infected with virus and the cells containing proteins expressed in the Baculovirus system are
the protein are harvested (Protocol 1.10). The more similar to those described in the bacterial expression
convenient way of producing large quantities of a system section.