Baculovirus System

Baculovirus Transfer Vector Plasmid Compatibility

A frequent customer query concerns baculovirus transfer vector plasmid compatibility with either flashBACTM or other systems for making recombinant viruses.  Obviously, if you are buying flashBACTM and our pOET range of transfer vectors you won’t have a problem.  However, given that baculovirus expression vectors have been around since 1983 and many labs have produced variations around a theme, it is not surprising that confusion can arise. Most baculovirus expression vectors

Baculovirus Transfer Vectors Compatible with flashBAC™

A question we are asked from time to time is to identify baculovirus transfer vectors compatible with our flashBAC™ system. We obviously nominate our own extensive range that is available but frequently someone has an old plasmid from years ago that they want to reuse. Originally they may have paired it with a linear DNA system such as BaculoGOLD or BacPAK6 or even wild type DNA where recombinant virus selection

Baculovirus-Expressed Secreted Recombinant Proteins

A question that often comes up from clients is what is the best signal peptide to use for baculovirus-expressed secreted recombinant proteins?  While we have discussed the best flashBAC™ variant to use for the production of secreted proteins in a previous blog, we have never considered the role of the signal peptide. In fact, while writing this blog, we realised that there was much that we didn’t know, so will

Transduction of Whole Porcine Kidneys with BacMAMs

One of the unexpected offshoots of the baculovirus expression system has been its development as a vehicle for the delivery of genes to mammalian cells.  These vectors are otherwise known as BacMams.  Although baculoviruses are unable to replicate in mammalian cells, they can enter them via receptor-mediated endocytosis.  In Frederick Boyce’s original paper the Rous sarcoma virus promoter was used to express beta-galactosidase in the HepG2 human liver cell line. 

P10 – the Forgotten Baculovirus Hyperexpressed Protein

The ubiquitous polyhedrin gene promoter, from which most baculovirus expression vectors are developed, has an oft forgotten rival – the p10 gene promoter.  The baculovirus expression system, on which our flashBAC™ vectors are based, derives from the original strategy whereby the virus polyhedrin gene coding region is replaced by the target recombinant gene sequences.  These sequences remain under the control of the polyhedrin gene promoter to obtain high levels of

Louis Pasteur and Insect Viruses

We told you last time about our trip to the Society for Invertebrate Pathology meeting in Tours, France.  This was another highly enjoyable gathering.  It also proved to be very successful for two of our OET-sponsored PhD students at Oxford Brookes University.  Mine Aksular won second prize for her poster presentation, “Improving baculovirus surface display system”. Leo Graves had a commendation for his talk, “3-Dimensional ultrastructural modelling of Autographa californica

Baculovirus Gene Mutations and Protein Expression

Have you ever wondered why the baculovirus we use for most protein expression purposes is called AcMNPV?  This is short for Autographa californica nucleopolyhedrovirus, which derives from the Latin name of the alfalfa looper, a pest of alfalfa crops.  The convention for naming baculoviruses is to use the host insect from which they were isolated.  This does mean that some viruses can effectively be named twice, if they are isolated

Longevity of Infectious Budded Baculovirus Stocks

A question that comes up repeatedly is the long term stability of recombinant budded baculovirus stocks.  This is a particular issue now that most of them are generated using insect cells grown in serum-free medium.  It has long been a mantra in virology that you should always have a little bit of protein in your virus suspension to help stabilise the virion structure or prevent non-specific binding to glass or

Titrating Stocks of Recombinant Baculoviruses using the Plaque Assay Technique

We were going to devote this blog to a comprehensive account of how you might determine the infectious titre of your virus stock.  However, when our CEO got wind of this he started ruminating about how things were “back in the day” and how he had to collect seaweed from the seashore to extract agar for his overlays…!  No, we didn’t believe him either, but he is approaching his 7th

Production of High Titre Virus Stocks

In our last blog post we talked about the process of transfecting insect cells with virus DNA and how you could recognize success or otherwise.  Transfections are normally done on a fairly modest scale (2-5ml) in culture dishes.  Therefore your P0 virus stock is of low volume and probably modest infectious titre (107 plaque forming units [pfu] per ml or lower; we will discuss how to measure this value in

Transfection of Insect Cells with Baculovirus DNA

Independent of the method you use to produce recombinant viruses, at some point you have to transfect insect cells with virus DNA to reconstitute infectious virus particles.  When using the flashBAC™ system, you simply add it to your transfer vector containing the foreign gene and use a suitable transfection agent (e.g. baculoFECTIN II )to achieve entry of DNA into the cells.  The transfected cells are then incubated at 28°C for