flashBAC™ by Oxford Expression Technologies Ltd

Oxford Expression Technologies Ltd Founded in 2007 as a spin out from Oxford Brookes University and the Natural Environment Research Council Technology based on insect baculovirus expression vectors (BEVs) Produces high levels of recombinant proteins in insect cells Ideal for producing complex, highly processed proteins and also virus-like particles (VLPs) Baculovirus vectors also used to transduce mammalian cells to express genes under (eg.) CMV promoter – The virus does not replicate in any mammalian cells 2

The flashBAC™ system (production of recombinant baculoviruses) flashBAC™ is based on homologous recombination between a replication deficient virus genome and a transfer vector with a foreign gene, which also rescues virus infectivity. 3 Only the recombinant virus with a restored ORF 1629 can replicate No need to plaque purify virus Represents a ONE-STEP process that can be used in high or low throughput applications flashBAC™ saves you time, simplifies the expression process and increases protein yield compared to other systems, as shown in the following slides

flashBAC™ time line comparison with other baculovirus systems 4 One step

Comparison of kinase production levels in flashBAC GOLD™ and Bac-to-Bac® expression systems The Western blots show a significant increase in kinase levels produced in the flashBAC GOLD™ system compared to Bac-to-Bac ®, at each time point recorded. This demonstrates that flashBAC GOLD™ is a favourable expression system when expressing certain proteins. (Hitchman RB el al Improved expression of secreted and membrane-targeted proteins in insect cells doi: /BA ) 5 ®

flashBAC™ variants (Available in kits for own use) flashBAC™ – Original, good for general purpose expression and BacMAM use flashBAC GOLD™ – Chitinase, cathepsin genes deleted, ideal for protein secretion flashBAC ULTRA™ – Also lacks p10 gene and other non-essential genes to enhance cell stability late in infection flashBAC PRIME™ – Essentially wild type genome, but superior for producing some VLP’s 6

flashBAC ULTRA™ flashBAC ULTRA™ has additional deletions to the non-essential genes p26, p10 and p74. These deletions increase cell stability and lower the burden on virus-infected cells, allowing more target protein to be produced. This combination makes flashBAC ULTRA™ the best platform for expression of membrane and cytoplasm targeted proteins. 7 EGFP Comparison of EGFP expression between the different flashBAC™ platforms. flashBAC ULTRA™ produces significantly higher yields of EGFP. (Hitchman et al. 2010, Genetic modification of a baculovirus vector for increased expression in insect cells, DOI /s y)

Advantages in flashBAC™ in human therapeutics and vaccines 8 Therapueutics and flashBAC™ - Use of BEVs in human therapeutics and vaccine work is set to expand greatly after the approval of baculovirus- derived Cervarix and Flublok, HPV and influenza vaccines (respectively). flashBAC™ uses homologous recombination unlike E.coli based systems (Bac-to-Bac®), which can retain residual antibiotic resistance markers and bacterial sequences, flashBAC™ would therefore be first choice for any therapeutic based protein production. ( Kwang et al Manufacturing of AcMNPV baculovirus vectors to enable gene therapy trials, Nature. doi: /mtm )

Summary flashBAC™ is a one-step process that: -Saves you time -Saves equipment -Simplifies protein expression -Decreases the chance of handling errors flashBAC™ has 4 variant platforms to suit your needs and optimise the expression levels of your protein flashBAC™ can be used in mammalian cell lines using BacMAM technology, ideal for producing virus-like particles (VLPs) Take advantage of the Gateway™ technology with our pOET Gateway™ transfer vectors OET is a name that you can trust for all things baculovirus

Apply now for a free transfer vector Once you start using our flashBAC ULTRA™ system, we know you’ll keep using it. Contact OET for a free 3 reaction flashBAC ULTRA™ kit when you purchase any pOET transfer vectors! Contact us on (00 44) or at Checkout our website