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 so that you can readily extract it using affinity chromatography. Other tags can also be used. You might also consider incorporating a protease cleavage site between the tag and the body of your protein so that you can separate the two sequences in a final purification step. Unlike many of the other operations in making recombinant baculoviruses, protein purification tends to require customized approaches, which are highly dependent on the target in question. Consequently, in this blog we offer some generic advice about protein purification based on our own experiences over the years with many different examples.
Another factor to consider is where your protein is going to end up after it is synthesized. Will it be nuclear, cytoplasmic or possibly secreted from the cells? Each destination presents its own unique challenges. The concentration of proteins that remain intracellular is initially very easy as virus-infected cells can be readily harvested using low speed centrifugation. Thereafter, things get a little more complicated as you have to decide upon a strategy for cell lysis and subsequent fractionation. Separating the nuclei from the cytoplasmic fraction can be achieved using non-ionic detergents and low speed centrifugation. However, you do have to be careful if your protein is sensitive to proteases as the wild type baculovirus encodes a very active cathepsin that can nibble away at your target. Using either flashBAC™ GOLD or ULTRA can be useful as both lack the cathepsin gene from the virus genome, which results in less degradation of the recombinant protein.
Removing the nuclei from a batch of virus-infected cells was certainly useful in a recent project we did for a customer. It cleaned up the protein preparation very nicely and by western blot the target was very homogeneous. Of course, you will need to optimize this step according to your scale of production. It is possible to saturate the process of cell lysis when using a non-ionic detergent such as Triton X-100 or NP40 so do some small scale purifications using the appropriate cell numbers and volumes. If you don’t then the cell fractionation process will not be 100% efficient.
Isolating a secreted protein from virus-infected cell culture medium presents a different problem. If you have several litres of starting material you can’t put this down an affinity column immediately. First, you will need to concentrate the protein from the medium using tangential flow chromatography. However, there is often a limit to how much you can concentrate the protein using this approach. As the volume is reduced its viscosity increases and it becomes harder and harder to remove liquid. Once this point is reached it can be useful to use polyethylene glycol to precipitate the proteins in the remaining volume of medium and thus quickly achieve a much greater degree of concentration. (You may also need to perform a buffer exchange for subsequent purification procedures). Of course, none of these steps do much to purify your protein, but they do reduce the volume that you have to work with.
If you are uncertain about protein purification, OET can do the whole process for you. If you are happy to purify the protein but struggle with processing large volumes of virus-infected cells we can do this bit for you and then send concentrated cells or cell culture medium to you for further processing. This is not as expensive as you might think. We always conduct a trial protein extraction from 1 litre of virus-infected cell culture before scaling up further, so you don’t need to commit to larger volumes right away.
Subsequently, depending on the nature of your protein you will probably employ some sort of affinity method to isolate the protein in a pure state.
One topic we have purposely left out of this blog is the isolation of virus-like particles (VLPs) from insect cells that have been made using recombinant baculoviruses. We will return to this in our next blog with an in depth consideration of all the factors you should think about when planning VLP production and purification.