Bac-to-Bac® Baculovirus Expression System From Invitrogen

Invitrogen has developed the Bac-to-Bac® Baculovirus Expression System for scientists who have chosen insect cells as their protein expression system. The basis of the system is that by creating a recombinant baculovirus that includes your gene of interest, you can infect insect cells, which will then (hopefully) produce your protein. In the Bac-to-Bac® System, Invitrogen have removed the need for homologous recombination of your gene with the baculovirus DNA, the main results being a reduction in overall time from cloning to protein and no need to plaque purify to get a single recombinant virus strain.

The process from gene to expressed protein is detailed in the system manual, and is summarized as follows: Your gene of interest is cloned into a donor plasmid, which is checked by restriction analysis, PCR or sequencing or a combination. The confirmed donor plasmid is transfected into special competent cells (supplied) which contain bacmid DNA and a helper plasmid which supplies Tn7 transposition functions which allow the transfer of your gene of interest into the bacmid DNA. The resulting large molecular weight recombinant bacmid DNA can be extracted and after PCR confirmation of insertion, may be transfected into insect cells to generate recombinant baculovirus. Once the virus is harvested and the titer checked by plaque assay, virus stocks can be grown and protein production assayed. As the system uses suspension cells, protein production can be scaled up as required.

The kit contains the basic items required for cloning and transfecting only: pFastBac™1 donor plasmid, pFastBac™1-GUS expression control plasmid, Max Efficiency® DH10Bac™ competent cells and Cellfectin® Transfection Reagent. Vector options, which must be purchased separately, include those with N-terminal histidine tags (HT Vectors) and a vector to express two proteins at once from different promotors (pFastBac™ Dual). A perusal of the manual shows several accessory products which must be purchased separately to complete the expression system. Invitrogen kindly provides a list of all the items with their catalog numbers, however, basic components such as antibiotics can be purchased from any reasonable supplier.

The Bac-to-Bac® Baculovirus Expression System has been our standard protein expression system for several years. We have successfully expressed proteins that range in size from 8 to 80 kDa. We have worked with membrane associated proteins (purified from the cells) and secreted proteins (purified from the supernatant). While the cloning and transpositions steps are relatively straightforward, an established tissue culture facility and experience with insect cell lines is recommended. We have successfully expressed a number of proteins and found that healthy, low-passage insect cells are essential for high levels of protein expression. Several insect lines are recommended for protein expression - Sf9, Sf21or High Five cells™, Mimic™Sf9, all from Invitrogen. We have used the Sf9 cells for several years, and found them satisfactory for our purposes, but it would be wise to check the cell line information to determine which line is best for your purposes. The high molecular weight bacmid DNA (+137kb) must be handled carefully to prevent shearing when extracted. We have found standard miniprep kits (Promega) are able to handle the large DNA by modifying the spin times and speed. Take care when storing the recombinant virus, as exposure to light or the incorrect temperature can cause the titer and subsequently, protein expression to drop.