Insoluble Protein – Inconsolable?

Published on October 13, 2016

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 of course!
The problem of an insoluble  protein is common to many expression systems, not just based on baculovirus expression vectors.  There is no easy answer.  You can isolate the protein under strongly denaturing conditions and then attempt refolding, but even that doesn’t always work.  We were musing on this issue recently after having the experience described in the first paragraph – although we did practice moderation in the bar!  The project remains ongoing, so we thought we would share our thought processes in deciding what to do next.  However, we can’t give specific details of the recombinant protein target owing to confidentiality agreements.
In wild type baculovirus-infected cells, most of you will know that two virus-encoded proteins (polyhedrin and P10) are synthesized to high levels.  These form the basis of the baculovirus expression vector system.  Their promoters, when linked with foreign coding regions, enable efficient production of recombinant proteins.  However, both polyhedrin and P10 proteins become insoluble very late in infection.  It is perhaps not surprising that high level accumulation of some recombinant products can also result in an insoluble protein.  Another baculovirus-encoded protein that becomes insoluble is the chitinase.  This is first synthesised in the late phase of virus gene expression (ca. 10h post infection onwards).
What is interesting about polyhedrin, P10 and chitinase is that initially, they are soluble proteins in virus-infected cells.  It seems that insolubility only develops as the protein concentration within virus-infected cells rises.  This may have relevance to the production of recombinant proteins that turn out to be insoluble.  Everyone optimises baculovirus expression to maximise yield.  However, it is just possible that by doing so you simply increase the likelihood of your protein being insoluble.
So what is our evidence to support this hypothesis?  Currently, absolutely none!  However, in the next few weeks we will be trying a variety of expression conditions in virus-infected cells to see if we can improve the recovery of the recombinant protein we mentioned above.  Our first test will be to see if harvesting the recombinant protein before it reaches its peak level in virus-infected cells improves solubility.  We will report back in future blogs on how we get on with this project.

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