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 cells secretory pathway.
flashBAC10TM builds upon this existing technology
by the removal of the P10 gene from the flashBACTM genome.
P10 is a 10 kDa protein, expressed concurrently with polyhedrin (polh) late
in infection and is nonessential in cell culture [2]. Both p10 and polh promoters
share a 12-nucleotide consensus sequence [3] containing the transcription initiation
ATAAG motif. P10 is activated a few hours before polh [4] and has been demonstrated
to compete with polh at a transcriptional level [5]. However, inhibition and
deletion of the p10 promoter has been shown to result in increased polh-controlled
protein production [2, 6] and polh mRNA levels [5]. P10 also associates with
occlusion bodies (OBs) [7] and is believed to mediate nuclear disintegration
late in infection as its disruption has been shown to prevent OB release [6].
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 [7] 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.
Advantages
- Increased recombinant protein transcription from the polh promoter
- More
efficient transport through the endoplasmic reticulum (ER)
- Increased cellular
stability and longevity
- Increased recombinant protein secretion
- Increased recombinant protein yield
- 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.
- 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.
For more information on commercial licences, please contact Helen Irving at
This e-mail address is being protected from spam bots, you need JavaScript enabled to view it
|
