OET are always happy and keen to help out and offer advice when projects are not running smoothly. Please always feel free to contact us and ask if there is anything we can do to help and/or assist you with.
Supply of transfer vector to clone your gene into a transfer plasmid yourself
Most transfer plasmids that are compatible with the transfer of the foreign genes into the virus genome by homologous replication are compatible with the flashBAC expression system and an up-to-date list can be found here. OET provides a number of its own proprietary transfer vectors (pOET series) that allow you to express genes under control of the very strong polyhedrin gene promoter or the p6.9 gene promoter, which whilst less strong than the polyhedrin gene promoter does express genes earlier in the virus infection cycle and can give higher overall yields of difficult to express proteins (membrane targeted or secreted).
In choosing a transfer vector, the following points should be considered. Alternatively, OET staff will be very pleased to provide advice on the best transfer plasmid to use for your project.
Promoter: Usually the polyhedrin gene provides the highest yields of recombinant protein. Sometimes, for membrane targeted or secreted proteins, using a late gene promoter (p6.9 for example) provides a higher overall yield of better quality protein. Dual promoter vectors can be used to express two genes simultaneously using one under the polyhedrin gene promoter and one under the p10 gene promoter, another very late gene.
Purification tags: If you wish to purify your protein, it is usually easiest to add a purification tag to either the C or N terminus of your protein. The most commonly used tag is 6xHis but baculovirus transfer plasmids with other tags are also available. You may also need to ensure that the transfer plasmid chosen includes a site to cleave the tag following purification.
Signal peptide sequence: Most reports indicate that almost all natural signal peptide sequences work in insect cells. However, if you wish to use an insect-specific signal peptide, transfer plasmids are available to add in-frame either a GP64 or chitinase signal peptide. After cloning the gene into your vector, we strongly recommend sequencing across the cloning 5’ junction to ensure that the sequence from the promoter into your foreign gene reads correctly (to ensure that the ATG start codon of your gene is the first to be encountered after the promoter sequence and that addition of any signal peptide/5’ purification tag is in frame).
Midi or maxi-prep material (10 µg min) should be sent to OET to make the recombinant baculovirus.