I'm interested in knowing how the yield and quality of recombinant protein compares between the two insect cell expression strategies.
Hi Neville,
Could you give a bit more information as to what you are looking to compare.
Normally transfection reagents such as GeneJuice would only be used at the very start of the baculovirus generation process to create the virus particles that would then be used for gene expression. So transfection is not generally used for protein expression.
However are you in fact asking for a comparison between using a plasmid with an IE promoter (e.g pIEx) for transient transfection and expression in insect cells against baculovirus? Or even Drosophila systems against Baculovirus?
In any case the most likely answer is : it depends upon the protein you are trying to express.
-Richard
Hi Richard,
Thanks for your interest. Yes, I mean the latter, "transient transfection and expression in insect cells against baculovirus".
Like most things there will indeed be protein-dependency for the results. However, I was wondering on average if yields (ug of protein/mL culture) are better using the transient transfection approach. My reasoning would be you avoid the toxicity inherent to using baculovirus infection, especially at high MOIs, (excluding any toxic effects of your protein of interest).
It seems like there is a trade off between culture volume and yield. For example, if the total expression is the same for each technique then it would be much cheaper to go down the baculovirus route. The main advantage I can see in this case is the time saved, 3-4 days vs 20 days. One could also make a stable insect cell line to save money on transfection reagents?
Thanks,
Nev
Hi Neville,
The couple of times I've used the pIEx system it's been to solve specific problems regarding the section of proteins from baculovirus infected insect cells. I both cases I seem to remember good levels of expression (This was quite some years ago now, so don't have figures) similar to what I'd expect from a fair to good BV expression.
As to whether to use this system instead of the baculoviral one? I really think that trying to second guess which will be the best expression system for your protein before hand, on what others have done is fraught with problems. That being said I do think it is a good expression system. As with other transient eukaryotic systems it is better for secreted proteins, and can have a distinct advantage over BVs in such cases.
There can be issues of scale-up, both in DNA requirements and Transfection reagents.
Yes it will be quicker on the first run, but if you know you will be repeating the production in BVs then scaling the virus production accordingly means resupply can be quick.
Producing stable lines will help, but then the time savings will be lost. As I recall this system uses a second plasmid for selection, which is not the most efficient for producing good expression clones, so more time spent finding them.
-Richard
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