Baculovirus Expression Systems and Services
Background and Advantages
Baculovirus-infected insect cells have been used to express thousands of recombinant proteins that include cytosolic, membrane-bound, and secreted proteins. Additionally, baculovirus expression systems are able to express human, mouse, plant, fungal, and bacterial proteins as well as a range of proteins derived from microbes. Since these cells are eukaryotic, they are known to express properly folded proteins, making baculovirus/insect cell expression the method of choice for proteins that will not fold correctly in other systems, such as those utilizing E. coli.
Genetic manipulations are performed in the virus and the genetically-altered baculovirus directs the expression of the heterologous protein during insect cell infection. This expression system uses a very strong polyhedron promoter in conjunction with powerful virus-encoded transcription machinery that expresses recombinant proteins at high levels during infection.
The advantages of baculovirus protein expression include:
- Endotoxin-free expression of desired protein(s);
- Significantly lower cost than other endotoxin-free (e.g., mammalian cell expression) systems;
- Improved solubility due to eukaryotic protein refolding;
- Compatibility with all known purification tags;
- Expression of high levels of secreted proteins;
- Eukaryotic intra-cellular transport and secretion;
- Post-translational modifications such a glycosylation and phosphorylation;
- Expression of multiple sub-unit protein complexes;
- Accommodation of large genes.
We offer a full range of custom baculovirus expression and purification services. To get started on a project or for more information, please contact us.
Baculovirus Expression Service Overview
|Gene Synthesis and Construct Assembly||Options:
1. Codon optimization followed by de novo gene synthesis and subcloning into a baculovirus transfer vector.
2. Customer supplies the completed transfer construct.
|Sequence verified construct||1-3 weeks|
1. Co-transfection: the transfer vector and baculovirus DNA are co-transfected into insect cells.
2. Bacmid: recombinant bacmid DNA is generated and transfected into insect cells.
|2 mL of P0 baculovirus stock (low titer)||1 week|
|Amplification||Insect cells are infected with P0 stock to amplify recombinant baculovirus titer.
Note: Although multiple amplification rounds are possible, the first round (P1 stock) is generally suitable for production.
|100 mL of P1 baculovirus stock (high titer)||1 week|
|Expression Testing||Insect cells are infected with amplified virus; culture samples taken at 48- and 72-hours post infection.||Experimental report with images of SDS-gels, blots, or pull-downs||1 week|
|Protein Production||Larger volumes of insect cell culture are infected with amplified virus; cells or media harvested at optimum time as determined by expression testing.||Cell pellets or media||1-2 weeks|
Baculovirus Protein Expression Service Details
Virus generation is the process of incorporating your gene into the virus genome. This starts with gene synthesis that includes codon optimization for protein expression in insect cells. Your gene will be subcloned into a baculovirus transfer vector followed by virus generation by either (1) direct transfection of a complete recombinant viral genome (i.e., bacmid) or (2) co-transfection of recombinant transfer vector and linearized baculovirus DNA. The newly generated baculovirus will be amplified to produce a high-titer stock for protein production.
The newly generated baculovirus (P0 stock) is not in sufficient quantity for use in protein production. This low titer stock is used to initiate further rounds of infection that yield a higher concentration and amount of baculovirus that is sufficient for protein production. Multiple rounds of virus amplification can yield enough baculovirus for 10 to 100 liters of production.
Expression Testing: Verification and Optimization
Measured amounts of amplified baculovirus are used to infect insect cells to verify expression of the target protein and determine the levels of expression at various times post infection. The usual times are 48 and 72 hours post infection but, in some cases, extended expression times are necessary. Our insect cells include Sf9, Sf21 and T.ni. In most cases, Sf9 cells are best for cellular proteins and T.ni cells are best suited for secreted proteins. Sf21 are always available as an alternative.
Time points (harvest points) will be processed for your protein by the most cost-effective method to determine the levels of expression in terms of milligrams per liter. Those methods of analysis include:
● Direct SDS-gel analysis of cell lysates or media;
● Affinity pull-downs of lysates or media;
● Western blot using target-specific antibodies.
Once protein expression has been verified with optimal harvest times, insect cell cultures are expanded to accommodate large scale production. A single round of amplification (i.e., 100 ml of P1 baculovirus stock) is generally sufficient to infect 10 liters of insect cells, which is well-suited for many applications. However, further rounds of amplification can be performed for larger scale or continuous protein production.