New Vector Increases Production and Quality for One-Step Baculovirus Protein
Expression.
Oxford Expression Technologies Ltd (OET), a leading provider of
baculovirus-based protein expression products and services, has introduced
flashBACULTRATM, a new baculovirus vector for increased yield and improved
quality of expressed protein.
Click here
to download the flashBACULTRA Press
Release (pdf).
Oxford Expression Techologies (OET) chose PepTalk 2008 in San Diego, California to launch flashBACGOLDTM, a baculovirus expression vector designed specifically to reduce proteolysis, maximise protein secretion and improve membrane protein targeting.
Click here to download the flashBACGOLDTM Press Release (pdf).
Cultured moth cells are the unlikely production platform for a technology that will aid drug discovery and help fight disease. The newly commercialised biotechnology company Oxford Expression Technologies Ltd (OET) is making this possible with an innovative range of products and services based upon the use of insect baculovirus protein expression technology.
Click here to download the OET Press Release (pdf).
OET announce the release of baculoQuant - a next-day baculovirus titration
service. The new service was officially launched at the Baculovirus Technology
Conference in Boston, U.S.A. on 25th September 2006.
A fast, accurate and efficient means of obtaining viral titres is essential
in the services we provide to pharmaceutical and biotechnology customers in
the area of insect cell expression and in our ongoing development of the baculoworkstation.
We have chosen to work with the quantitative Polymerase Chain Reaction (QPCR)
viral titration service from Oxford Expression Technologies as this fulfils
these criteria with knowledgeable, personable service.
Dr. K S Richards, Senior Scientist, NextGen Sciences Ltd.
OET has developed a rapid baculovirus titration method (1) using quantitative
real-time PCR (QPCR) and Taqman fluorogenic probes, specifically designed
to the viral genome. Taqman technology takes advantage of the 5 exonuclease
activity of Taq polymerase to digest a probe, hybridised between flanking PCR
primers, and labelled with two fluorescent dyes (2). The dyes undergo fluorescent
resonance energy transfer(FRET) because of their proximity to each other (3).
Cleavage by Taq during primer elongation interrupts the FRET and allows the
reporter dye to fluoresce for every cycle of the PCR. This increase in fluorescence
can be analysed in real-time and allows quantification during the exponential
phase of the reaction. OET has utilised this technology to develop an accurate
and rapid baculoviral titration system. The system has been calibrated against
known viral titres, previously determined by plaque assay, and has been semi-automated
using a Corbet Research liquid handler to prepare the QPCR reactions ready
for quantification on an ABI 7500 Sequence Detection System.
References
- Hitchman RB, Siaterli EA, Nixon CP, King LA. 2006. Quantitative real-time
PCR for rapid and accurate titration of recombinant baculovirus particles.
Biotechnology and Bioengineering.
- Holland PM, Abramson RD, Watson R, Gelfand
DH. 1991. Detection of specific polymerase chain reaction product by utilizing
the 5'3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc.
Natl. Acad. Sci. U.S.A. 88: 72767280.
- 3. Heid CA, Stevens J, Livak KJ, Williams
PM. 1996. Real time quantitative PCR. Genome Res. 6: 986994.
Although many proteins can be produced to high levels using standard baculovirus
expression vectors, membrane-targeted and secreted proteins have often given
relatively poorer yields. The reason for this is not completely understood
but has been attributed to virus infection compromising the function of the
insect cell secretory pathway.
Studies in the insect virus research group at Oxford Brookes University have
led to the discovery that a virus-specific enzyme, chitinase, is targeted to
the cell endoplasmic reticulum (ER) where it accumulates and forms a quasi-crystalline
array. The ER becomes vesiculated and almost unrecognisable towards the end
of the virus replication cycle. Chitinase itself is not essential for virus
replication in cell culture, its role is to mediate the degradation of chitin
in the larval cuticle, promoting characteristic terminal insect liquefaction.
At OET we have produced a baculovirus expression vector in which the chitinase
gene has been deleted from the virus genome. Virus replication, production
of recombinant virus and function of the polyhedrin gene promoter (controlling
foreign gene expression) are not affected by this deletion. Tests with a wide
range of secreted and membrane-targeted proteins indicate, however, that this
chitinase-deficient expression vector can increase the yield of the protein
in the culture medium (or plasma membrane) by up to 20-fold (range 2-fold to
20-fold). Generally, use of this vector does not appear to enhance the yield
of recombinant proteins targeted to the cytoplasmic or nuclear compartments.
Oxford Expression Technologies have announced a working partnership with
ECACC (the European Collection of Cell Cultures) to offer a combined baculovirus
expression service supporting scaled up production. ECACC's Bioprocessing Group
specialises in developing scaled up animal cell culture processes. ECACC's
expertise in scaling up to volume production compliments OET's leading position
in baculovirus expression techniques. Together, ECACC and OET can offer a complete
service with the capability to produce recombinant protein in quantities from
1L to multiples of 100L.
Contact ECACC for more details.
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