1. Clone the gene of interest into the appropriate baculovirus expression vector and cotransfect with linearized baculoviral DNA
(available from various vendors) or use transposition to create recombinant bacmid DNA in E. coli (BAC-TO-BAC system; Invitrogen). Alternatively, purify circular wild-type baculovirus DNA.
2. Cotransfect baculovirus DNA with the recombinant baculovirus plasmid or transfect purified bacmid DNA into Sf 9 insect cells.
3. Collect the medium, which contains the baculoviral particles, and plaque the virus on Sf 9 cells to separate recombinant from nonrecombinant virus.
This and subsequent rounds of plaque purification are optional when linearized virus that contains a lethal deletion is used (e.g., BaculoGold from Pharmingen), as in that case >99.9% of all
amplified virus particles will be recombinant because of selection pressure.
Similarly, when bacmid DNA is used, no purification is required.
4.Amplify the virus stock by infecting fresh insect cells and determine titer of the amplified virus stock (UNIT 16.10, Basic Protocol 4).
5.Express the protein of interest by infecting a new batch of insect cells with the hightiter baculovirus stock.
Determine the expression level of the recombinant protein of interest and analyze its biological activity.
Related readingļ¼
Introduction To Baculovirus Expression System
Metabolic Labeling Of Recombinant Proteins-Baculovirus Expression System