Insect cell/BEVS expression technologies
There are a number of different technologies that can be used to generate a recombinant baculovirus containing the gene of interest. Some of the most commonly used technologies are listed below. Screening protein expression using multiple baculovirus technologies can determine the most suitable for producing your target protein.
System |
Transfer Vectors |
Features |
Bac-to-BacTM |
pFastbac-1,
pFastBac Dual |
Requires integration with baculovirus shuttle vector (Bacmid) in bacterial host cell before transfection |
flashBACTM |
pBac-1 |
Transfection with linear DNA, recombination occurs within host insect cells. Deletion of chiA enables better secretion of recombinant protein |
BaculoGOLDTM |
pVL1392, 1393 |
Transfection with linear DNA, recombination occurs within host insect cells |
BaculoDirectTM |
pENTR |
Transfer vector recombines with linear DNA before transfection into host cell. Requires Ganciclovir as selection agent |
Cell lines
The most commonly used cell lines are Sf9 and High FiveTM (see table below). Both can be grown as adherent or suspension cultures and scaled up from microplates to shake flasks and rocking motion bioreactors. They are also able to be grown in serum-free media (SFM) and do not require the addition of CO2 gas. Some commonly used insect cell lines are listed in the table below.
Selection of the right cell line is critical for successful protein production and PEF recommends screening at least two insect cell lines. Plasmids, strains and cells are available from the UQ resource centre.
Cell Line |
Origin |
Features |
Sf9 |
Spodoptera frugiperda |
Adapted to SFM, commonly used to express all types of recombinant proteins |
Mimic Sf9 |
Spodoptera frugiperda |
Adapted to SFM, expresses mammalian glycosyltransferases to generate proteins with more complex N-glycan structures |
High FiveTM |
Trichoplusia ni |
Adapted to SFM, commonly used for secretion of recombinant proteins |
Growth temperature
Although insect cells are typically cultured at 27°C, lowering the temperature (e.g. to 21˚C) can improve protein folding and target protein production.
Expression duration
The timeframe of protein expression can also play a role in successful target protein production. For example, protein harvested 48 hours post infection (hpi) may have a different expression profile to protein harvested at 72 hpi. Screening protein expression at multiple time points may determine the optimal condition for your target protein.