Baculovirus Protein Expression

Producing and Maintaining AcNPV-derived Baculoviruses

Introduction

The use of insect cells and lytic baculoviruses for expression of full-length mammalian proteins has been the recent method of choice for many disciplines. Autographa californica nuclear polyhedrosis virus (AcNPV) infects the clonal tissue culture line, Sf9, derived from Spodoptera frugiperda insect cells. Expression of the highly abundant polyhedrin gene is non-essential in tissue culture and its strong promoter can be used for the synthesis of foreign gene products. The polyhedrin promoter is maximally expressed very late in infection when the lytic virus is already killing the host cells, giving a reasonable chance for high levels of expression even for certain toxic proteins. Many post-translational modifications are made correctly in insect cells and proteins unable to be expressed in E. coli have been successfully expressed in the insect cell system.

The BD BaculoGold™ Linearized Baculovirus DNA from BD Biosciences provides a tool for recombination efficiencies close to 100%. The principle of this technique lies in the construction of a modified type of baculovirus DNA, which contains a lethal deletion. This DNA does not code for viable virus. Only co-transfection of insect cells with the viral DNA and a complementing transfer vector construct reconstitutes viable virus. Essentially, 99% of all virus plaques are derived from plasmid-rescued viruses which contain and express the foreign gene from the plasmid. Since the BD BaculoGold Linearized Baculovirus DNA contains a lacZ gene that is replaced by recombination with the plasmid containing the foreign gene, all recombinants will produce colorless plaques on X-gal plates. The small portion of non-recombinant virus plaques (usually less than 1%) will stain blue on X-gal plates. If preferred, the virus may be amplified from a single plaque from a plaque assay.

Selection of a Transfer Vector

All polyhedrin gene locus-based baculovirus transfer vectors can be used to rescue the lethality of the BD BaculoGold DNA. Transfer vectors from BD Biosciences Pharmingen include pVL1392, pVL1393 (provided with the BD BaculoGold Transfection Kit), pAcSG2, pAcGP67, pAcUW21, pAcUW51, pAcMP2 and pAcMP3, and pAcGHLT (GST-fusion vectors) and pAcHLT (6xHis-fusion vectors). For a complete listing and specific information about these vectors, please visit our website at www.bdbiosciences.com. Other vectors may also be used, but have not been tested for compatibility with the BD BaculoGold Linearized DNA.

Recommended Controls

When performing a co-transfection to produce recombinant virus, it is important to include both positive and negative controls. These controls are used in all applications for producing and maintaining recombinant baculovirus. When using the BD BaculoGold™ Linearized Baculovirus DNA, recombinant baculovirus expressing the XylE protein can be used as a positive control and are generated by performing a co-transfection with the pVL1329-XylE Baculovirus Control Plasmid, Cat. No. 554807. Cells infected with this recombinant virus express recombinant XylE and turn yellow in the presence of the substrate catechol. Alternatively, recombinant virus can be generated using the BD BaculoGold Bright Linearized Baculovirus DNA which contains the GFP gene, expressed constitutively. The GFP protein is fluorescent and cells generating virus expressing this protein will glow green and can be assayed or sorted using flow cytometry or visualized by fluorescence microscopy. Recombinant virus can be titered or purified using these applications. Uninfected Sf9 cells are used as the negative control. Uninfected cells continue to proliferate; therefore, relative cell number is a criterium for degree of infection. The use of both positive and negative controls is particularly valuable for the end-point dilution and plaque assays.

Material Required

Materials and Reagents

Transfection Reagents:

BD BaculoGold Transfection Kit, Cat. No. 560129, contains:

Linearized Baculovirus DNA 2.5 µg
pVL1392 Baculovirus Transfer Vector 5 µg
pVL1393 Baculovirus Transfer Vector 5 µg
pVL1392-XylE Baculovirus Control Vector* 5 µg
Transfection Buffer A 5 ml
Transfection Buffer B 5 ml

or

BD BaculoGold Linearized Baculovirus DNA, Cat. No. 554739
Transfection Buffer A&B Set, Cat. No. 554806
Transfer Vector of choice

TNM-FH Insect Cell Medium, Cat. No. 554760
Sf9 cells in TNM-FH Medium, Cat. No. 554763 (grow to log-phase, at least 98% viable)
1 ml, 5 ml, 10 ml, 25 ml and/or 50 ml sterile pipets
1.5 ml sterile microcentrifuge tube (or comparable)
20 µl and 1000 µl sterile pipet tips
50 ml sterile conical tube, BD Falcon™ Cat. No. 352098
15 ml sterile conical tube, BD Falcon™ Cat. No. 352097
Tissue Culture Dish, 60 x 15 mm, BD Falcon™ Cat. No. 353802
Tissue Culture Dish, 150 x 25 mm, BD Falcon™ Cat. No. 352097
*Catechol, Sigma, Cat. No. C-9510 (When using pVL1392-XylE Control Vector)

Equipment

  • Sterile laminar flow hood
  • Electric pipettor
  • L 2, L 20 and L 1000 Pipettors
  • Mini centrifuge
  • 27°C Incubator
  • Light microscope