flashBACGOLDTM Enhanced yields for difficult to express proteins
Baculovirus genomes contain several auxiliary genes, which are non-essential
for replication in insect cell culture. Two of these are chitinase (chiA),
which encodes an enzyme with exo- and endochitinase activity6 and a cathepsin-like
cysteine protease (v-cath)7. In an infected insect, chitinase and cathepsin
facilitate host cuticle breakdown and tissue liquefaction at the very late
stages of infection, so releasing the virus to infect more hosts.
Confocal
and electron microscopy observations of insect cells infected with AcMNPV have
shown that chitinase is targeted to the endoplasmic reticulum (ER), where it
is densely packed in a para-crystalline array, blocking and severely compromising
the function and efficacy of the secretory pathway9. V-cath accumulates in
the ER at early times post-infection as an inactive proenzyme (pro-v-cath)
and is then activated by proteolytic cleavage upon cell death, but is sensitive
to the cysteine protease inhibitor E-6410.
It has optimum activity at pH 5.0 -
5.5, although it also shows measurable activity up to pH 7.0 11. Chitinase
may act as a chaperone for the proper folding of pro-v-cath in the ER12. Together
these enzymes compete with the recombinant protein for limiting cellular resources,
putting a huge burden on the protein translocational machinery13. As a protease,
v-cath will also degrade susceptible recombinant proteins, particularly in
the later stages of infection when the polh promoter is most active.
The deletion of both chiA and v-cath from flashBACGOLDTM has
improved the efficacy of the secretory pathway and resulted in a greatly enhanced
yield of recombinant proteins that are secreted or membrane targeted (in comparison
to recombinant viruses that encode chiA and v-cath). Results also show a significant
reduction in degradation of protease-sensitive targets and increased production
and stability of some intra-cellular proteins (manuscript in preparation).
Complex secretory or membrane-bound glycoproteins are often more difficult
to express using baculovirus and produced in lower amounts compared to cytoplasmic
or nuclear proteins 1,2,3,4,5.
flashBACGOLDTM is a baculovirus
expression vector that has been designed to reduce proteolysis, maximise
protein secretion and improve membrane protein targeting and is based on
our patented flashBACTM system, removing the necessity
for plaque-purification.
As such, it is also back-compatible with
all existing baculovirus transfer vectors based on homologous recombination
in insect cells at the polyhedrin locus.
- Kaba SA, Salcedo AM, Wafula PO, Vlak JM, van Oers
MM. (2004) Development of a chitinase and v-cathepsin negative bacmid for
improved integrity of secreted recombinant proteins. J Virol Methods. 122,
113-8.
- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA. (1993)
Influence of different signal peptides and prosequences on expression and
secretion of human tissue plasminogen activator in the baculovirus system.
J Biol Chem. 268, 16754-62.
- Kost TA, Condreay JP (1999). Recombinant baculoviruses
as expression vectors for insect and mammalian cells. Curr Opin Biotechnol.
10, 428-33. Review.
- Tomiya N, Betenbaugh MJ, Lee YC (2003). Humanization
of lepidopteran insect-cell-produced glycoproteins. Acc Chem Res. 36, 613-20.
Review.
- van Oers MM, Thomas AA, Moormann RJ, Vlak JM. Secretory
pathway limits the enhanced expression of classical swine fever virus E2
glycoprotein in insect cells (2001). J Biotechnol. 86, 31-8.
- Hawtin, R. E.,
Arnold, K., Ayres, M. D., Zanotto, P. M. d. A., Howard, S. C., Gooday, G.
W., Chappell, L. H., Kitts, P. A., King, L. A., and Possee, R. D. (1995).
Identification and Preliminary Characterization of a Chitinase Gene in the
Autographa californica Nuclear Polyhedrosis Virus Genome. Virology 12, 673-685.
- Slack,
J. M., Kuzio, J. & Faulkner, P. (1995). Characterization
of v-cath, a cathepsin L-like proteinase expressed by the baculovirus Autographa
californica multiple nuclear polyhedrosis virus. Journal of General Virology
76, 1091-1098.
- Hawtin, R. E., Thomas, C. A., Gooday, G. W., Kuzio, J. A.,
King, L. A. and Possee, R. D. (1997). Liquefaction of Autographa californica
nucleopolyhedrovirus-infected cells is dependent on the integrity of virus-encoded
chitinase and cathepsin genes. Virology, 238, 243-253.
- Thomas, C. A., Hawes,
C. R., Lee, B. Y., Min, M-K, King, L. A. and Possee, R. D. (1998). Localisation
of a baculovirus-induced chitinase in the insect cell endoplasmic reticulum.
J Virol. 72, 10207-10212.
- Hom, L. G., Ohkawa, T., Trudeau, D. and Volkman,
L. E. (2002) Autographa californica M nucleopolyhedrovirus Pro-V-CATH is
activated during infected cell death, Virology 296, pp. 212–218.
- Bromme,
D. & Okamoto, K. (1995) The baculovirus cysteine
protease has a cathepsin B-like S2-subsite specificity. Biol Chem Hoppe Seyler.
10, 611-5.
- Hom, L. G. & Volkman, L. E. (2000). Autographa californica
M nucleopolyhedrovirus chiA is required for processing of V-CATH. Virology.
277, 178-83
- Possee, R. D., Thomas, C. J. & King, L. A. (1999).
The use of baculovirus vectors for the production of membrane proteins in
insect cells. Biochem. Soc. Transcr. 27, 928-932.
For more information on commercial licences, please contact Helen Irving at
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