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
1. 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.
2. 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: 7276?7280.
3. Heid CA, Stevens J, Livak KJ, Williams PM. 1996. Real time quantitative PCR. Genome Res. 6: 986?994.

OET announces improved production of secreted and membrane-targeted proteins

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.

OET Announces Partnership with ECACC

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.