Protein Production With Recombinant Baculoviruses in Lepidopteran Larvae

Abstract

SummaryWith an increasing need for functional analysis of proteins, there is a growing demand for fast and cost-effective production of biologically active eukaryotic proteins. The baculovirus expression vector system is widely used, and in the vast majority of cases cultured insect cells have been the host of choice. A low cost alternative to bioreactorbased protein production exists in the use of live insect larvae as "mini bioreactors." In this chapter, we focus on Trichoplusia ni as the host insect for recombinant protein production, and explore three different methods of virus administration to the larvae. The first method is labor-intensive, as extracellular virus is injected into each larva, whereas the second lends itself to infection of large numbers of larvae via oral inoculation. While these first two methods require cultured insect cells for the generation of recombinant virus, the third relies on transfection of larvae with recombinant viral DNA and does not require cultured insect cells as an intermediate stage. We suggest that smallto mid-scale recombinant protein production (mg-g level) can be achieved in T. ni larvae with relative ease.

Functional and translational analyses of a beta-glucosidase gene (glycosyl hydrolase family 1) isolated from the gut of the lower termite Reticulitermes flavipes

“…The inserted RfBGluc-1 gene was confirmed by sequencing and used for bacmid preparation and transfection into Sf9 cells, which were then incubated at 27 C for 4 days (Passage 0). The cell pellet from Passage 0 was tested by Western blotting with anti-His antibody to confirm expression of His-tagged protein, and recombinant virus was harvested and injected to T. ni larvae as described previously (Liu et al, 2007;Kovaleva et al, 2009). Larvae were orally infected with active pre-occluded baculovirus (Liu et al, 2007;Kovaleva et al, 2009), harvested in large scale, and stored at À80 C for later processing.…”

“…The cell pellet from Passage 0 was tested by Western blotting with anti-His antibody to confirm expression of His-tagged protein, and recombinant virus was harvested and injected to T. ni larvae as described previously (Liu et al, 2007;Kovaleva et al, 2009). Larvae were orally infected with active pre-occluded baculovirus (Liu et al, 2007;Kovaleva et al, 2009), harvested in large scale, and stored at À80 C for later processing. Recombinant protein was recovered with greater than 98% purity from T. ni homogenates by (1) centrifugation, (2) 0.22 mM supernatant filtration, (3) tandem Ni-IMAC (nickel-immobilized metal affinity chromatography), and (4) Sephadex G-25 chromatography for buffer exchange.…”

Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

“…The cell pellet from Passage 0 was tested by Western blotting with anti-His antibody to confirm expression of His-tagged protein. After 2 days, the recombinant virus from Passage 1 was harvested and injected to T. ni larvae as described previously (Liu et al, 2007;Kovaleva et al, 2009). Larvae were orally infected with active pre-occluded baculovirus (Liu et al, 2007;Kovaleva et al, 2009), harvested in large scale, and stored at À801C for later processing.…”

“…After 2 days, the recombinant virus from Passage 1 was harvested and injected to T. ni larvae as described previously (Liu et al, 2007;Kovaleva et al, 2009). Larvae were orally infected with active pre-occluded baculovirus (Liu et al, 2007;Kovaleva et al, 2009), harvested in large scale, and stored at À801C for later processing. Recombinant protein was recovered from clarified T. ni homogenates by tandem Ni-IMAC (nickel-immobilized metal affinity chromatography) followed by buffer exchange with Sephadex G-25 chromatography.…”

Production of a recombinant antibody fragment in whole insect larvae

“…One approach to circumvent the current limitations associated with insect cell culture is to use whole insect larvae as the biological unit of protein production [17,22,32]. We have developed technology for automated mass rearing and oral infection of millions of larvae with recombinant baculoviruses [31].…”