Baculovirus Expression Systems
Baculovirus Expression Systems
DNA Transfection for Baculovirus Expression Vector System
Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector (donor or shuttle) plasmid DNA containing the foreign gene to be expressed and BaculoGold™ DNA (PharMingen), Bac-N-Blue™ DNA (Invitrogen), or BacPAK6™ DNA (Clontech). Alternatively, insect cells are transfected with a recombinant bacmid DNA constructed by transposition of the donor plasmid DNA in E. coli cells, the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. The baculovirus DNA is from Autographica californica nuclear polyhedrosis virus (AcNPV).
The BaculoGold™ and Bac-to-Bac™ methods are designed to achieve virtually 100% recombination efficiencies and recombinant protein expression is subsequently evaluated using recombinant virus amplified (without plaque purification) in P2 in insect cells. A single plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) or higher. Recombinant baculovirus derived from all other commercially available baculovirus DNA preparations is produced with 80-90% efficiency and requires plaque purification to remove parental virus.
Recombinant Baculovirus Stocks
High titer P2 virus stock is produced from the P1 virus (the original virus from cell culture supernatant of co-transfected cells) in Sf9 cells. P2 virus stock is generally produced in 100 ml of serum-free medium. A P3 virus (50 ml or 500 ml) is produced by infecting Sf9 cells with a P2 virus at a low multiplicity of infection (MOI=0.1).
Plaque Assay and Purification of Recombinant Baculovirus
Plaque assays are done by infecting sf9 or sf21 insect cells with the P1 virus (or higher passage virus) at a low multiplicity of infection (MOI = 0.1) and overlaying the infected cells with agarose. Well isolated plaques are scored and virus is subsequently amplified in monolayer cultures of Sf9 cells prior to preparation of larger volume high-titer stocks.
Time-Course Study of Protein Expression
100 ml suspension cultures of insect cells are infected with a high-titer baculovirus stock at an MOI=1-2. Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection to evaluate the integrity, stability and optimum yield of the product(s) of gene expression. The results can be used to determine optimal conditions for protein production. Harvested samples are analyzed for expression of the recombinant protein by western blot. The staff can compare recombinant protein production in at least three different cell lines (sf9, sf21, and High Five) if necessary.
Large-Scale Production of Recombinant Proteins
High density (1-2 x106 cells/ml) suspension cultures of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells are infected at a multiplicity of infection (MOI) equal to 1-2. High Five cells are preferred for production of secreted proteins. We routinely infect cultures (250 ml to 1 L) of insect cells in serum-free medium containing Pluronic F-68 (to prevent cell damage due to shearing) in spinner bottles.
For additional information about insect cell culturing and baculovirus expression systems, please see:
Guide to baculovirus expression systems and insect cell culturing (PDF)
Bac-to-Bac Baculovirus expression systems (PDF)
Baculodirect Baculovirus Expression Systems (PDF)
Baculovirus Expression Vector Systems (PDF)
Bac-N-Blue Baculovirus Expression System (PDF)