Editing Baculovirus Expression (section)

== Preparing and titering baculovirus working stocks for protein production. ==

<br>Because we can only send you a small sample of a plaque-purified, first passage (PP1P1) virus stock, you will need to produce and titer a working virus stock before producing your recombinant protein of interest. You should grow the virus by infecting Sf9 or Sf21 cells cultivated in TN-MFH medium containing 10% FCS and 0.1% pluronic F68 (“complete” TN-MFH). Please NOTE that baculovirus stock produced in serum-free media are not stable. For more detailed protocols, see (Jarvis, 2011; Murhammer, 2007; O'Reilly, Miller, and Luckow, 1992), among others.

=== A) Amplify the PP1P1 stock: ===

<br>1) Aseptically seed 0.5 X 106 Sf9 cells/ml of complete TN-MFH medium into a shake flask.

<br>2) Incubate overnight in a shaking incubator at 28oC, 125 rpm.

<br>3) Aseptically add ~0.1 mL of the recombinant baculovirus PP1P1 stock.

<br>4) Incubate in a shaking incubator at 28oC until you see clear signs of infection:<br>a) Look for swollen cells, swollen nuclei.<br>b) This usually takes ~3-5 days.

<br>5) Aseptically harvest the medium and pellet cells and cell debris:<br>a) Ten min at top speed (~1000 g) in a tabletop centrifuge.

<br>6) Aseptically remove the supernatant, transfer to a fresh tube or bottle:<br>a) This is the PP1P2 virus stock.<br>b) Wrap with aluminum foil to protect from light and store at 40C (Jarvis and Garcia, 1994).<br>(i) These stocks will slowly lose titer during storage (~1 log/yr; Jarvis and Garcia, 1994)).<br>(ii) Can freeze aliquots for longer-term storage/archiving.

=== B) Titer the working virus stock: ===

<br>1) Aseptically seed 0.75 X 10<sup>6</sup> Sf9 cells/well into one or more 6-well plates:<br>a) Cells will stick best if pelleted, resuspended in serum-free TN-MFH.<br>b) Use 3 ml per well for seeding.<br>c) Allow cells to adhere for ~1 h at 28<sup>o</sup>C.

<br>2) Aseptically prepare a 10-fold dilution series (10-1 to 10-6) with PP1P2 stock:<br>a) Use complete TNM-FH as the diluent.<br>b) Can typically plate (10-4, 10-5, 10-6) and get a useful range of plaques to count.

<br>3) Drain medium from cells.

<br>4) Replace with 2 mL of diluted virus in each well:<br>a) Plate each dilution in duplicate.<br>b) Incubate for ~1 h at 28<sup>o</sup>C.

<br>5) Prepare agarose overlay:<br>a) Melt 1.5 mL of 2% agarose/well in a microwave oven.<br>b) Equilibrate in a 60oC water bath.<br>c) Warm 1.5 mL of complete 2X Grace’s medium/well in an 30oC water bath.

<br>6) Overlay the infected cell monolayers:<br>a) Aseptically mix equal volumes of agarose and complete 2X Grace’s medium (3.0 mL/well).<br>b) Mix thoroughly.<br>c) Aseptically remove the virus from each well in the plaque assay.<br>d) Overlay with 3 mL per well.<br>e) Allow agarose to harden for 10 min.

<br>7) Seal in a plastic baggie and incubate upside down for 7-10 days at 28oC.

<br>8) Remove from baggie, count plaques on upside down plate under a dissecting microscope.<br>a) Titer = avg # plaques X reciprocal of the dilution (pfu/mL).