Editing Baculovirus Expression

== 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).

== <br>II. Protein production and purification. ==

=== A) Infect insect cells to produce the protein of interest. ===

<br>1) Aseptically seed 0.5 X 106 Sf9 cells/ml in your favorite insect cell medium into a shake flask:<br>a) Best to use Sf9 cells adapted to a serum-free medium to purify a secreted protein.

<br>2) Incubate in the shaking incubator overnight at 28oC.

<br>3) Perform a viable cell count to determine cell density (should ~double overnight).

<br>4) Aseptically add the volume of virus needed to achieve a multiplicity of infection of 2-5 pfu/cell.

<br>5) Incubate in a shaker-incubator at 28oC for 2-3 days:<br>a) Typically, 48-60 h.<br>b) But, best to perform preliminary experiments to determine the time of infection that yields the highest levels of intact product for each recombinant protein.

<br>6) Harvest the medium and pellet cells and cell debris:<br>a) Ten min at top speed (~1000 xg) in a tabletop centrifuge.

<br>7) Remove the supernatant, place in a fresh tube or bottle as the cell-free fraction.<br>a) For secreted proteins, process the supernatant as the source of recombinant protein.<br>b) For intracellular proteins, process the cellular pellet as the source of recombinant protein.

<br>8) For detailed information on immobilized metal affinity chromatography, see:<br>a) (Crowe et al., 1994).<br>b) (Petty, 2001).<br>c) Vendors instructions.<br>d) Many papers on HIS-tagged proteins expressed in baculovirus-infected insect cells.

=== B) Metal affinity-purify the protein of interest (secreted proteins). ===

<br>1) Dialyze the supernatant against dialysis buffer (50 mM NaH2PO4, 500 mM NaCl; pH 8.0) for 12-<br>18 h with a buffer change after 6 h.

<br>2) Wash Probond (Invitrogen) or your favorite metal affinity resin with dialysis buffer.

<br>3) Incubate the dialyzed supernatant with the washed resin at RT for 3 h with gentle agitation.

<br>4) Transfer into an empty column barrel and allow the resin to settle into a bed.

<br>5) Wash with 5 column volumes of dialysis buffer containing low concentration of imidazole:<br>a) We use 20-50 mM imidazole for washes.

<br>6) Elute with 1 column volume of dialysis buffer containing high concentration of imidazole:<br>a) We use 250 mM imidazole.

<br>7) Assess purification by analysis of starting material, unbound, wash, and elution fractions.

=== <br>C) Metal affinity-purify the protein of interest (intracellular proteins). ===

<br>1) Wash the infected cells once or twice with ice cold TBS (50 mM Tris, 100 mM NaCl; pH 8.0).

<br>2) Lyse the cells in one of two ways:<br>a) Resuspend pellet in a minimal volume of ice-cold dialysis buffer; freeze-thaw twice.<br>b) Resuspend pellet in a minimal volume of TBS + 1% NP40); incubate for 10 min on ice.

<br>3) Shear DNA by tituration with an 18-gauge needle until the solution is no longer viscous.

<br>4) Centrifuge for 15 min at ~10,000 xg at 4oC.

<br>5) Harvest the supernatant and use for metal affinity chromatography as described above (D2).

== III. References. ==

<br>Crowe, J., Dobeli, H., Gentz, R., Hochuli, E., Stuber, D., and Henco, K. (1994). 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression/purification. Methods Mol Biol 31, 371-87.<br>Jarvis, D. L. (2011). Recombinant protein expression in baculovirus-infected insect cells. In "Methods Navigator Protocols (online)". Elsevier, Amsterdam.<br>Jarvis, D. L., and Garcia, A., Jr. (1994). Long-term stability of baculoviruses stored under various conditions. Biotechniques 16(3), 508-513.<br>Murhammer, D. W. (2007). "Baculovirus and insect cell expression protocols." 2nd ed. Methods in Molecular Biology, 388 Humana Press.<br>O'Reilly, D. R., Miller, L. K., and Luckow, V. A. (1992). "Baculovirus expression vectors." W.H. Freeman and Company, New York.<br>Petty, K. J. (2001). Metal-chelate affinity chromatography. Curr Protoc Mol Biol Chapter 10, Unit 10 11B.<br><br>

[[Category:Protocols]]