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December 01, 2009 (Vol. 29, No. 21)

Novel Vectors for Baculovirus Expression

Gene Cloning and Protein Expression in Insect Cells Is Faciliatated by New Methodologies

  • 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.

  • Polyhistidine Fusion Proteins

    Click Image To Enlarge +
    Figure 1. New pFastBac/TOPO vectors for intracellular expression

    The topoisomerase-adapted vectors pFastBac/NT-TOPO and pFastBac/CT-TOPO (Figure 1A) allow the production of N-terminal and C-terminal intracellular polyhistidine fusion proteins. Blunt-ended PCR products can be cloned with an efficiency >95%. To test the expression performance of the vectors, several human genes were cloned and eventually expressed in SF21 cells. All the proteins tested could be readily detected as sharp bands with the expected molecular weight in Western blots. It is worth noting that higher yields were consistently observed in those samples derived from pFastBac/NT-TOPO.

    A subset of the samples was subjected to TEV protease cleavage. Even when lysates, rather than purified proteins, were employed, protease digestion resulted in virtual completion in only 30 minutes of incubation (Figure 1B).

    To further validate the performance of these new type of vectors, a notoriously difficult-to-express protein, the serotonin GPCR 5HT1a was cloned into pFastBac/CT-TOPO and the resulting viral particles were used to infect High Five cells. The 5HT1a protein localized primarily to the surface of the cells, and the protein was properly folded as suggested by the corresponding ligand binding assay (Figure 1C).

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