I'm looking to start expressing & purifying some proteins via the
baculovirus system. I've never done this before and so I thought someone
experienced with this system might be able to answer a few questions.
1) Which system works the best and is the most cost-efficient? A friend
of mine uses the Pharmingen system and is happy with it. From what I
understand, the limiting and most costly reagent is the linear baculovirus
DNA, which costs about $300 for 5 transfections (we already have access
to the rest of the reagents, including insect cells). I've also been
looking at this system from Gibco called Bac-to-Bac. It does all the
necessary recombination in bacteria rather than insect cells and so
doesn't require the linear baculo DNA. However, their kit costs almost
$500 and also limits you to 5 transfections. From what I understand, the
limiting reagent in their kit are the competent bacteria that contain the
baculo DNA, but it seems to me that one should be able to grow more of
these bugs and thus make as many different proteins as you want from this
single kit. Anyone know anything about this?
2) The proteins I'm most interested in producing are transmembrane
glycoproteins that are highly modified by glycosylation and disulfide
bonds. Which of the various baculovirus promoters should I use? I've
read that while the very late promoters like polyhedron give you more
yield, the late promoters like the basic protein promoter and the 39k
protein promoter give you better post-translational modifications and may
be a better choice for proteins that are highly modified. Also, would
there be any benefit to using baculovirus signal peptides rather that WT
signal peptides for this type of protein?
Thanks in advance for any feedback at all on these questions. I hope I
can return the favor in the future.
Vince Solomon
Microbiology & Molecular Genetics
Harvard Med School
--
