Insect Cell/BEVS Expression - General Info - Protein Expression Facility

The insect cell/baculovirus expression vector system (BEVS) is becoming increasingly popular for the production of recombinant proteins.

The system offers a number of advantages, including:

Ability to perform complex post-translational modifications (PTMs)
High success rate of soluble protein recovery
Suitable for the production of large protein complexes, e.g. virus like particles (VLPs)
High protein expression levels compared to other higher eukaryotes

One of the main drawbacks of the system is longer timeframes for protein expression in comparison to other host systems.

How do I optimise my protein expression in the baculovirus/insect cell system?

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-Bac^TM	pFastbac-1, pFastBac Dual	Requires integration with baculovirus shuttle vector (Bacmid) in bacterial host cell before transfection
flashBAC^TM	pBac-1	Transfection with linear DNA, recombination occurs within host insect cells. Deletion of chiA enables better secretion of recombinant protein
BaculoGOLD^TM	pVL1392, 1393	Transfection with linear DNA, recombination occurs within host insect cells
BaculoDirect^TM	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 Five^TM (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 Five^TM	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.