Since the pioneering interferon expression studies conducted in the early 1980s, baculovirus-based expression in insect cells has become one of the most widely used systems for production of recombinant proteins. Several unique features account for the popularity of this approach.
- It is a eukaryotic expression system, therefore it uses many of the protein modification, processing, and transport pathways present in higher eukaryotic cells.
- High expression levels (up to 1 g of protein product/L of culture) can be obtained, without many of the hassles encountered when using mammalian cell cultures.
- The viral genome can accommodate relatively large fragments of foreign DNA.
- The virus can be propagated to high titers without the need of a helper DNA.
- Availability of suspension cell lines (many of them adapted to grow in serum-free conditions) makes the system amenable to scale-up.
- As part of the baculoviridae family, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is safe to use as it is noninfectious to vertebrates.
- A wide variety of transfer vectors have been developed that made the recombinant virus isolation a simple process.
A variety of approaches to transfer genes from standard vectors into the viral genome have been developed. Efforts to ease the requirement for a plaque assay resulted in the design of a bacterial transposition method marketed under the name of Bac-to-Bac®, from Invitrogen, part of Life Technologies. Concurrently, an in vivo recombination method between a transfer vector and a restricted viral DNA was developed.
The approach, which restores the function of an essential gene, was further improved to include a blue vs. white plaque visualization method, and it is currently commercialized under the name of Bac-N-Blue™.
More recently an in vitro recombination system has been developed. In this system (commercially available under the trademark of BaculoDirect™) the foreign gene is transferred from an entry clone to the viral DNA via Gateway LR recombination. The thymidine kinase gene present in the Gateway® cassette acts as a strong counter selectable marker when cells are grown in the presence of ganciclovir.
In this article, we present three new pFastBac™/TOPO® vectors, compatible with the Bac-to-Bac system, that allow the expression of poly-histidine protein fusions either cell-associated or secreted into the medium. We also generated a Gateway-compatible BaculoDirect genome that enables the expression of intracellular N-terminal GST protein fusions.