Recombinant Protein Production via BVES

Recombinant Protein Production via Baculovirus Expression Systems (BVES)

Baculovirus expression systems (BVES) represent an alternative approach to produce large amounts of properly folded and functional recombinant proteins. The benefits of protein expression with baculovirus include the virus being able to accommodate large inserts, eukaryotic post-translational modification, enhanced protein folding and function, high expression levels, easy scale up with high-density suspension culture, and safety. The Facility has >20 years of experience in preparing baculovirus vectors from nearly all commercially available BVES. View a list of available vectors.

Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors, have facility staff engineer the plasmid DNA construct, or to provide the facility with small volumes of previously prepared virus stock.

Available services

Preparation of high-titer baculovirus stock

The generation of recombinant baculovirus is achieved via one of two approaches. Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector plasmid DNA containing the foreign gene to be expressed and baculovirus DNA from Autographica californica nuclear polyhedrosis virus (AcNPV)-Baculogold method. Alternatively, insect cells are transfected with a recombinant bacmid DNA recovered by transposition of the transfer vector DNA in E. coli cells (DH10Bac), the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. Investigators have the option to provide the facility with previously constructed baculovirus transfer vectors or have facility staff engineer the plasmid DNA construct. View more information about baculovirus expression systems.

Baculogold (Pharmingen, Sigma)-3 weeks

  • Transfection of insect cells (Sf9) with the recombinant transfer vector (containing gene of interest to be expressed) and baculoviral DNA.

  • Amplification of high-titer recombinant baculovirus stock (250 ml-P2).

  • (Optional) virus titering.

Bac-to-Bac (Invitrogen)-3 weeks

  • Transposition of pFastBac transfer vector in DH10Bac, Bacmid preparation, PCR comfirmation of bacmid transposition.

  • Transfection of insect cells (Sf9) with Bacmid DNA.

  • Amplification of high-titer recombinant baculovirus stock (250 ml-P2).

  • (Optional) virus titering.

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 round of plaque purification of recombinant virus from the initial virus production (P1 virus) is optional before virus is amplified in a second passage (P2) for these methods. 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.

Timeline:

~3 weeks

Required Materials:

5 µg of recombinant baculovirus transfer plasmid containing gene of interest.

Deliverables:

250 ml of high-titer (≥1 x 10pfu/ml) recombinant baculovirus stock solution in serum free medium ready to express the protein of interest

Amplification of high-titer baculovirus stocks

 The purpose of this service is to prepare high-titer (>108 pfu/ml) baculovirus stocks in serum free medium for preparative scale productions of recombinant protein in insect cells. Titration of the final high-titer stock is an optional service.

Timeline:

2~3 weeks

Required Materials:

At least 5 ml of recombinant baculovirus stock.

Deliverables:

Specified quantity (in ml) of high-titer recombinant baculovirus stock solution ready to express the protein of interest.

Analytical scale productions for optimization of protein expression

The purpose of this service is to aid investigators in assessing the optimal conditions for protein production. The results can be used to determine optimal conditions for protein production. There are numerous examples where the integrity, stability and biological activity of recombinant proteins vary with time after infection. Therefore the recommendation of the facility is that each new baculovirus for a protein be extensively characterized in analytical scale productions before proceeding to large-scale productions.

This service entails the infection of a 100 ml suspension culture of upto three insect cell lines [Sf21, Sf9, and High Five (T.ni)] using a high-titer baculovirus stock. Cells or conditioned media (for secreted proteins) are harvested at 24, 48, and 72 hours post infection. Harvested samples are analyzed for expression of the recombinant protein by western blot. Investigators can complete the Western blot analysis or have the facility staff complete the analysis for an additional fee (customer must provide appropriate gene specific antibodies, if desired).

Timeline:

1 week

Required Materials:

At least 5-10 ml of high-titer recombinant baculovirus stock.

Deliverables:

Cell pellets (or conditioned medium for secreted proteins) from 10-20 ml of infected insect cells ready for expression/solubility analysis by western blotting or small scale purification. A sample of uninfected insect cells is also provided as a negative control.

Preparative scale productions

Expression of recombinant protein under optimized conditions in high-density (1-2 x106 cells/ml) suspension cultures (50 ml-1L) of Sf9, Sf21, or Trichoplusia ni (commonly referred to as High Five) cells at a multiplicity of infection (MOI) equal to 1-2. We recommend use of High Five cells for production of secreted proteins.

Timeline:

1-2 weeks

Required Materials:

Predetermined, optimized protein expression conditions (cell line, MOI, infection time).

 

1 liter: at least 25 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

2 liter: at least 50 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

4 liter: at least 100 ml of high-titer (>10pfu/ml) recombinant baculovirus stock.

Deliverables*:

Cell pellets ready for protein purification or in the case of a secreted recombinant protein, the customer will receive the supernatant

*We do not guarantee the yield of purified protein/volume of culture since it changes from protein to protein. To achieve a certain yield, additional culture volumes may be required.

**We offer discounts for multi-liter productions done simultaneously.