Resources

Healthy Culture of Insect Cells

Following on from our last blog on the revival of cells stored in liquid nitrogen, we now provide some tips on the continued healthy culture of insect cells.  Whether you are maintaining cells as monolayer or suspension (shake or spinner) culture you will probably faithfully record your name/medium used/date of sub culture/passage number on the side of your new flask.  Alternatively, instead of using a new flask you may simply

Cryogenic Storage and Revival of Insect Cells

Cryogenic storage of cells is essential to ensure long term, convenient access to new stocks. How well this process is conducted can have a significant impact on the revival process too. In this article we provide a few tips on how both freezing and revival of insect cells can be successfully accomplished.

Insoluble Protein – Inconsolable?

You may have had the experience!  An expression project is going really well.  You inserted your gene into a baculovirus vector (preferably one of our flashBAC™ range), amplified the virus, tested expression, obtained a rewarding blob of stained protein on a gel, went on to purify it – only to find an insoluble protein in your hands.  Cue to retire to the bar for a consolation beer – in moderation

Protein Expression and Directed Mutations

If you want to make a modified form of a protein as a recombinant product, this can have unintended consequences on its synthesis in your chosen expression system. Even minor changes to a sequence can affect structure, rendering it difficult to produce or unstable with the result that protein yields are severely reduced. Avoiding this problem may be difficult, particularly if you want to study a particular domain of a protein via mutagenesis. However, including appropriate controls, such as an unmodified version of the protein can be very useful in troubleshooting these problems. Even changes to gene codon usage, while not affecting protein sequence, may affect secondary mRNA structure and thus reduce the translation.

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

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