Superior Expression and Yield
flashBACTM is an expression system that allows the production of multiple recombinant
viruses in a one-step process [1]. This technology has greatly de-skilled the
process of making recombinant viruses and increased the throughput of recombinant
protein production. It also has a chitinase gene deletion (chiA) that greatly
improves the movement of recombinant proteins through the cellular secretory
pathway.
flashBACGOLDTM is a further improvement on this system,
by the removal of both chiA and a protease (v-cath) from the virus genome [2].
These modifications are primarily designed to facilitate the movement of recombinant
proteins through the cell’s secretory pathway and to prevent their degradation
once they are released into the culture media.
flashBACULTRATM has
taken this technology a step further, by the removal of three more virus genes
(p10, p74 and p26) from the flashBACULTRATM genome.
P10
is a 10 kDa protein, expressed concurrently with polyhedrin (polh) late in
infection and is nonessential in cell culture [3]. Both p10 and polh promoters
share a 12-nucleotide consensus sequence [4] containing the transcription initiation
ATAAG motif. P10 is activated a few hours before polh [5] and has been demonstrated
to compete with polh at a transcriptional level [6]. However, inhibition and
deletion of the p10 promoter has been shown to result in increased polh-controlled
protein production [3, 7] and polh mRNA levels [6]. P10 also associates with
occlusion bodies (OBs) [8] and is believed to mediate nuclear disintegration
late in infection as its disruption has been shown to prevent OB release [7].
Recent work has also shown that it forms extensive cytoskeleton-associated
or cytoskeletal-like structures in the nucleus and cytoplasm, potentially de-stabilizing
the cells cytoskeleton [8] and further depleting cellular resources.
Deletion
of p10 increases polh activity providing more recombinant protein, increases
nuclear and cellular stability, ensuring a longer timeframe for protein expression
and removes a major competitor for limiting cellular resources.
P74 is non-essential in cell culture but is essential for oral infectivity
of occlusion-derived virus (ODV) in the host where it plays a role in midgut
attachment and fusion [9]. Deletion of p74 has been shown to have no effect
on virus production in vitro [10].
Deletion of p74 further increases the biosafety profile of recombinant baculoviruses
in the environment, making them unable to traverse the insect gut wall.
P26 is an early gene that codes for a 240-amino acid polypeptide of unknown
function [11] and has the same 5′ terminus as p10 [12]. Deletion of the 3’-end
of p26 and fusion to lacZ or p10 have previously been shown to have no effect
on virus replication in vitro [6, 13].
Deletion of p10, p74 and p26 removes an unnecessary genetic burden from the
recombinant virus genome, providing a more efficient baculovirus expression
vector.
Advantages
- Increased recombinant protein transcription from the polh promoter
- More
efficient transport through the endoplasmic reticulum (ER) and secretory
pathway
- Increased cellular stability and longevity
- Increased recombinant protein
secretion
- Increased recombinant protein yield
- Reduced proteolysis and degradation
- Increased recombinant protein quality
- Possee RD , R.H., KS Richards, SG Mann, E Siaterli, CP Nixon, H Irving,
R Assenberg, D Alderton, R J Owens, L A King Generation of baculovirus vectors
for the high throughput production of proteins in insect cells. Biotechnology
and Bioengineering, 2008.
- Hitchman, R., High-level recombinant protein expression
in insect cells using a chitinase and cathepsin deletion deletion-virus BMC
Biotechnology, 2008.
- Vlak, J.M., et al., Functional studies on the p10 gene
of Autographa californica nuclear polyhedrosis virus using a recombinant
expressing a p10-beta-galactosidase fusion gene. J Gen Virol, 1988. 69 (
Pt 4): p. 765-76.
- Rohrmann, G.F., Polyhedrin structure. J Gen Virol, 1986.
67 ( Pt 8): p. 1499-513.
- Roelvink, P.W., et al., Dissimilar expression of
Autographa californica multiple nucleocapsid nuclear polyhedrosis virus polyhedrin
and p10 genes. J Gen Virol, 1992. 73 ( Pt 6): p. 1481-9.
- Chaabihi, H., et al., Competition
between baculovirus polyhedrin and p10 gene expression during infection of
insect cells. J Virol, 1993. 67(5): p. 2664-71.
- Williams, G.V., et al., A
cytopathological investigation of Autographa californica nuclear polyhedrosis
virus p10 gene function using insertion/deletion mutants. J Gen Virol, 1989.
70 ( Pt 1): p. 187-202.
- Carpentier, D.C., C.M. Griffiths, and L.A. King, The
baculovirus P10 protein of Autographa californica nucleopolyhedrovirus forms
two distinct cytoskeletal-like structures and associates with polyhedral
occlusion bodies during infection. Virology, 2008. 371(2): p. 278-91.
- Faulkner,
P., et al., Analysis of p74, a PDV envelope protein of Autographa californica
nucleopolyhedrovirus required for occlusion body infectivity in vivo. J Gen
Virol, 1997. 78 ( Pt 12): p. 3091-100.
- Kuzio, J., R. Jaques, and P. Faulkner,
Identification of p74, a gene essential for virulence of baculovirus occlusion
bodies. Virology, 1989. 173(2): p. 759-63.
- Liu, A., et al., Nucleotide sequence
of a portion of the Autographa californica nuclear polyhedrosis virus genome
containing the EcoRI site-rich region (hr5) and an open reading frame just
5' of the p10 gene. J Gen Virol, 1986. 67 ( Pt 11): p. 2565-70.
- Rankin, C.,
B.F. Ladin, and R.F. Weaver, Physical mapping of temporally regulated, overlapping
transcripts in the region of the 10K protein gene in Autographa californica
nuclear polyhedrosis virus. J Virol, 1986. 57(1): p. 18-27.
- Rodems, S.M. and
P.D. Friesen, The hr5 transcriptional enhancer stimulates early expression
from the Autographa californica nuclear polyhedrosis virus genome but is
not required for virus replication. J Virol, 1993. 67(10): p. 5776-85.
For more information on commercial licences, please contact Helen Irving at
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