Resources

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

Producing Virus-Like Particles (VLPs) in Insect Cells

Using baculovirus expression vectors to produce virus-like particles (VLPs) is not new.  Intact, non-infectious poliovirus1 and bluetongue virus2 VLPs were synthesized using some of the earliest baculovirus vectors with considerable success.  In many of the projects we undertake for customers we have also generated VLPs for a wide range of different viruses.  They are ideal candidates for use as sub unit vaccines.  The immunogenic parts of the virus can be

Producing Secreted Proteins

We had promised to consider the production of virus like particles in this blog, but have postponed this topic to a later date after the subject of optimal secretion of recombinant proteins came up at a recent meeting we attended.  It seems some of you are reporting poor yields of secreted protein using baculovirus vectors after preliminary studies of transient expression in insect cells suggest they should be much higher. 

Protein Purification

After scaling up recombinant baculovirus-insect cell cultures (blog) we now come to what can be a very tricky part of the operation, namely how do you extract your protein?  Hopefully, this process was in your mind before you even started cloning your gene of interest into a transfer vector.  For most recombinant proteins it can be convenient to add a string of histidine residues to either end of your target

Scaling up Protein Production

Once you have decided on the optimal conditions for producing your recombinant protein target in baculovirus-infected insect cells, your thoughts may turn to scaling up its production prior to purification.  You may be fortunate enough that the yield of recombinant protein is very high and your requirements fairly modest that only a small volume of virus-infected cells is required.  On the other hand, you may have lofty ambitions for your

Optimising Protein Expression

Once you have finished making your recombinant baculoviruses expression vector you naturally want to maximize the amount of protein made in insect cells.  A point we have emphasized in a previous blog is the importance of determining the infectious titre of your virus stock accurately.  It is also prudent that you produce a stock of virus large enough that you can conduct a series of experiments to optimise protein production

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

Rapid Virus Titration by QPCR

Knowing the infectious titre of a recombinant virus stock is really important prior to testing for protein production in insect cells.  It enables you to add the optimal quantity of virus to initiate virus infection.  Too much virus and you are wasting your stock.  Too little virus and you won’t establish a synchronous infection.  For some proteins this may not matter, as the virus produced by the initial round of

Virus Like Particles VLPs for Therapeutics and Research

Oxford Expression Technologies (OET) is now able to apply its flashBAC™ baculovirus expression system to vaccine production, particularly in the exciting new field of virus like particle (VLP) vaccines. A virus like particle is an assembly of virus structural proteins that mimics the configuration of a real virus, except that it contains no genetic material. If a person is vaccinated with VLPs then an immune response is generated as if

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

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