Page 25 - Macromolecular Crystallography

14 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 1.9 Amplification of the recombinant Baculovirus virus stock

Materials 3. Incubate the plate at 27 C for 3 days.
◦
Sf 9 cell culture 4. Examine the plate for signs of infection after 2 days of
150-mm tissue culture plates incubation using a microscope.
Recombinant Baculovirus low titre viral stock 5. Collect the virus supernatant and remove cell debris by
centrifugation at 10,000 g for 5 min at 4 C.
◦
Insect cell medium
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6. Store at 4 C in a dark place or cover the tube with a
Microcentrifuge tubes
piece of aluminium foil.
Microscope
7. Determine the recombinant viral titre by plaque assay.
Method 8. Amplify two or three times to obtain a high titre stock by
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1. Pipette 2.1 × 10 cells onto a plate and allow the cells repeating Steps 1 to 5.
◦
to form a monolayer by placing the plate on a level surface 9. Store the virus stock at 4 C in a dark place for up to
◦
for 40–45 min at room temperature or 27 C. 6 months or at –80 C for longer storage periods.
◦
2. Add 100 µl of the recombinant virus stock, keeping the
multiplicity of infection below one.
Protocol 1.10 Expression of the recombinant protein in the Baculovirus system in
monolayer cultures

Materials 4. Add high-titre virus stock to the plates so that
Sf 9 cell culture multiplicity of infection is between 3 and 10.
◦
150-mm tissue culture plates 5. Transfer the plates to 27 C incubator and leave
High-titre recombinant viral stock for 3 days.
6. Examine the plates for signs of infection using a light
EX-CEL 405™serum-free medium containing 50 µg/ml
microscope.
gentamycin sulphate, 2.5 µg/ml amphotericin B, and
7. Harvest the supernatant and cells from the plates and
10% fetal calf serum
pellet the cells at 1000 g for 5–10 min at room temperature.
◦
27 C incubator
Secreted proteins are found in the supernatant whereas
Microscope non-secreted proteins remain in the pellet.
Haemocytometer 8. Store the pellets and supernatants at –80 C.
◦
Trypan Blue Stain (0.2%) solution in PBS 9. If the recombinant protein is in the cell pellet,
resuspend the pellet in an appropriate lysis buffer
Method containing inhibitors of proteases.
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1. Pipette 2.0 × 10 cells onto each of the several plates 10. Purify the target protein using the same methods as
and allow the cells to form a monolayer by placing the plate described for purification of proteins expressed in the
on a level surface. bacterial system.
2. Add fresh medium to a final volume of 30 ml in each 11. If the recombinant protein is secreted it will be present
plate without disturbing the cell monolayer. in the supernatant. In that case, add an equal volume of an
3. Calculate the amount of virus stock required using appropriate buffer, containing inhibitors of proteases, to the
the equation:
supernatant and proceed with the purification of the target
ml of virus required = multiplicity of infection (plaque protein as described for the bacterial system.
forming units/cell) × number of
cells/titre of virus per ml.