Editing Baculovirus Expression (section)

=== 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.