Oxford Expression Technologies Ltd

Transcription

1 Oxford Expression Technologies Ltd Founded in 2007 as a spin out from Oxford Brookes University and Natural Environment Research Council Technology based on the insect baculovirus expression vectors (BEVs) Produces high levels of recombinant proteins in insect cell culture Ideal for producing complex, highly processed proteins Also ideal for producing 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 1

2 Proud to be market leaders Oxford Expression Technologies Ltd (OET) was founded by two key opinion leaders, Profs R. Possee and L. King OET is one of the few companies still expanding and developing the baculovirus system OET, always happy to answer technical questions and trouble shoot any aspect of BEVs 2

3 Why use recombinant baculovirus expression systems There are several advantages to using the baculovirus/insect cell protein expression system: High protein expression levels Superior post translational modification (PTM) compared to bacteria and yeast cells, similar to those in mammalian cells Easy to handle and scale up Cost effective Applications include functional assays, protein interactions, antibody generation and therapeutics 3

4 OET Products flashbac vector kits for protein expression in: Insect cells Single, multiple proteins and multi subunit formation (e.g. VLP s) Mammalian cells Single, multiple proteins and multi subunit formation (e.g. VLP s) Accessory products Insect cells Transfer vectors Transfection reagents Cell culture media Virus titration kits (QPCR) 4

5 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. 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 really does save time, simplifies the whole expression process and increases protein yield compared to other systems, as shown in the following slides 5

6 flashbac variants (Available in kits for own use) flashbac Original, good for general purpose expression 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 flashbac HT Variant producing virus stocks with enhanced infectious titres, particularly useful as a BacMAM for mammalian cell transduction 6

7 flashbac flashbac contains a deletion to the chitinase gene from the baculovirus backbone. Removal of chitinase gene improves intracellular recombinant protein transport Chitinase has been shown to localise around the ER where it is densely packed in a paracrystalline array, which severely compromises the secretory pathway. Confocal imaging shows that deletion of chitinase decreases build up of secreted protein (urokinase) around the ER, which improves the secretory pathway and therefore protein yields. 7

8 flashbac GOLD flashbac GOLD has a deletion to the cathepsin gene from the baculovirus backbone, in addition to the chitinase deletion. Removal of cathepsin gene improves stability/reduces recombinant protein degradation Together these deletions increase the levels of harvestable target protein. Levels of harvested membrane targeted glycoproteins are greatly increased by reducing protein degradation caused by cathepsin. (Hitchman et el Improved expression of secreted and membrane-targeted proteins in insect cells doi: /ba ) 8

9 flashbac ULTRA EGFP Comparison of EGFP expression between the different flashbac platforms. flashbac ULTRA produces significantly higher yields of EGFP. (Hitchman et al Genetic modification of a baculovirus vector for increased expression in insect cells, DOI /s y) 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. 9

10 flashbac PRIME OET has developed flashbac PRIME, which is essentially wild-type baculovirus. This platform has proven very effective when expressing certain virus-like particles (VLPs) that do not bud from the cell membrane, such as circovirus (as shown in the micrograph images). flashbac PRIME takes advantage of the natural lytic process that occurs during the virus-infected cells cycle, which releases the protein into the supernatant, allowing easy purification of the target protein. flashbac ULTRA flashbac PRIME Electron microscopy images of semi-purified of circovirus VLP, produced using recombinant flashbacultra or flashbacprime viruses. 100 nm scale bars. 10

11 flashbac time line comparison with other baculovirus systems 11

12 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 ) 12

13 OET is the one-stop shop for baculovirus protein expression In addition to OET s patented flashbac technology, we also provide a range of products that are specifically chosen for their excellence in recombinant baculovirus protein expression flashfectin and baculofectin II transfection reagents, both specifically designed for insect cell transfections Range of poet transfer vectors, including the new poet Gateway vectors (utilise Life Technologies Gateway system) and BacMAM vectors that enable transduction of genes into mammalian cells Expression Systems insect media, both serum and serum free Insect cells Sf9, Super Sf9 and Sf21 cells lines baculoquant all-in-one viral quantification/titration kits, enables easy and accurate viral titres to be measured 13

14 poet transfer vectors flashbac is compatible with most baculovirus transfer vectors, making the transition to flashbac as easy as possible. OET has a range of transfer vectors for customers to choose from: Transfer vectors containing polyhedrin gene promoter with multiple cloning sites (MCS) His tagged vectors Gateway vectors for easy and efficient gene shuttling BacMAM transfer vectors that enable transduction of target genes into mammalian cells safety and efficiently For more, please see the OET price/product list 14

15 Production of recombinant BacMAMs flashbac can be used to make expression vectors specific for mammalian cell transduction. The construction method is essentially the same but utilises a transfer vector with a copy of the CMV promoter for foreign gene expression. The BacMAM system is particularly useful to researchers aiming to produce protein with the highest level of PTMs, especially in vaccine and therapeutic based work. With baculovirus derived vaccines now on the market, this is an area that seems set to grow rapidly. A. Mock transduced HEK293 cells B. BacMAM EGFP transduced HEK293 cells C. BacMAM EGFP transduced HEK293 cells with added sodium butyrate All cells observed at 24 h post transduction 15

16 OET Ltd Contract Services A flexible, bespoke service for production of recombinant proteins From gene sequence to purified protein, or any point in between Protein expression offered in insect or mammalian cell culture 16

17 Summary The recombinant baculovirus expression system is able to produce high quality, well folded proteins at a competitive cost OET have made significant improvements to original baculovirus expression systems in the flashbac range There are currently 4 different flashbac variants that enable you to choose the platform best suited for your needs OET provides everything you need to produce your recombinant proteins, from insect cells, insect culture media to our baculoquant virus titration kit OET has a good and well established name in the protein expression industry We are always happy to help and offer advice 17