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 plastic vessels. When all insect cell culture relied on the use of medium supplemented with bovine serum to aid growth this was not a problem. Now that we mostly use serum-free media, it is good practice to add bovine serum to a concentration of at least 2% after harvest of the virus stock. Some prefer to add this when the cell culture is originally inoculated with virus.
Of course, the presence of bovine serum in the virus stock may be undesirable for some purposes. In this event it is important to realise that the infectivity of the budded virus will decline quite rapidly – possible losing 90% over three months. This can be a particular problem when using baculovirus vectors that retain the virus-encoded cysteine protease or cathepsin gene.
To explain this further, it is necessary to consider the replication of baculoviruses in insect cells. Baculovirus genes are expressed in three major phases: early, late and very late. Moving swiftly past the early one, the late phase sees production of infectious virus particles, which bud from the plasma membrane. This phase also sees the production of the virus-encoded cathepsin. In nature, this protein plays a part with other proteins in liquefaction or melting of virus-infected insects to help release occlusion bodies in the terminal stages of replication. However, when using the baculovirus expression system in cell culture, it has the annoying side effect of affecting the stability of both protease-sensitive recombinant proteins and also the infectivity of virus stored in the absence of bovine serum.
Fortunately, if you use either flashBAC GOLD or flashBAC ULTRA, both of these virus DNAs lack the cathepsin gene. This means that recombinant viruses prepared using either system will not produce the virus-encoded protease that can degrade both your target protein and reduce virus infectivity on prolonged storage at 4°C.
You might wonder why a virus protease produced in the late phase of virus gene expression should be such a problem, given that the very late phase is when recombinant protein is synthesized, usually under the polyhedrin gene promoter control. However, the three phases of baculovirus gene expression overlap, with some early genes such as immediate early gene 1 expressed throughout infection. Late gene expression kicks in at about 8 hours post infection (hpi) and continues until at least 24 hpi. Very late gene expression initiates from about 15 hpi while the cell is still pumping out budded virus and producing the cathepsin, which is also quite stable. Therefore, protease activity is rampart throughout most of the late/very late infection phases. We could go into a lot more detail about its role in the native virus infection in insects, but that would be straying too far from the use of baculoviruses as expression vectors.
Once you have your virus stock(s) cryostorage at -70/80°C seems to preserve infectivity very well. We haven’t found any need to snap freeze budded virus stocks using dry ice/ethanol or liquid nitrogen, a procedure recommended for many enveloped viruses such as influenza. However, it won’t do any harm. If you have a need to use a particular recombinant virus on a regular basis it is efficient to freeze aliquots, thaw one for titration purposes and then assume that samples thawed subsequently retain the same infectious titre. Tests we have done show that a single freeze/thaw cycle does not affect the infectious titre significantly. It should be feasible, therefore, to store a reasonable volume of virus for several months and access it for protein production purposes as and when required.
Over the last few months we have covered a whole range of topics relevant to cell culture and generating recombinant viruses with defined infectious titres. It is probably about time to get to the sharp end of using these reagents for their intended purpose, namely the production of recombinant protein! Therefore, in our next blog we will start to address the various factors you should consider when optimization protein production by baculoviruses in insect cells.