Baculovirus expression systems - which one??
dk at no.email.thankstospam.net
Tue Feb 5 07:18:59 EST 2002
"Anthea Scothern" <anthea.scothern at umist.ac.uk> wrote:
>I am trying to decide between the Bac-to-Bac from what was Gibco, BacnBlue
>from Invitrogen (both now available in the Invitrogen catalogue), Clontech's
>Bac Pak and Novagen's BacVector system - after a day of reading catalogues,
>I can't handle any more BacAnything.
>Does anyone have any recent experience with any of these kits/vectors/viral
>DNA? My archive search only shows discussions a couple of years old.
>I've been using an inherited combination of Baculogold DNA with BacPak
>vectors and my virus stocks are very unreliable - sometimes expression,
>sometimes not; so I think it's time to reassess and reclone. Any comments
>would be gratefully received.
If you already have the virus of interest, it's not worth it to redo the whole
thing. Simply do plaque purification, pick several clones, amplify all once,
analyse cells left after amplification, choose one that gives highest
expression levels and amplify it further. If after doing this you continue
to have problems, you are doing something else wrong.
I've used Gibco's system extensively. It works well and is very easy.
Some specific things about it:
None of the various cell lines I tried grows in Gibco's serum-free
medium. Adapting them is possible but not worth it if you are working
with non-secreted protein.
7-8 hours transfection before changing medium to complete with
serum works better than suggested 5 hours.
Expression levels tend to drop considerably with every round of
amplification. I attribute this to production of some of non-expressing
viruses right after transfection. Over time these guys take over and
even though your titers are great there is little expression. Two ways
to cope with it. One is to do plaque purification. I find that once the
virus is cloned, I can do several rounds of amplification while maintaning
high expression levels. Note that in theory this defeats the purpose of
Bac-to-Bac kit, which aims to completely eliminate the need to do
plaque picking. Another is to severely limit amplification rounds. Thsi is
what I do most of the time: transfect 10 cm dish with the whole bacmid
miniprep, let cells get really sick (usually takes at least 4 days) and
amplify this initial stock only once (200 ul transfection sup/150 cm dish
for as long as takes for cells to become very, very sick; usually 3-4 days
depending on how successful your transfection was). This gives
you 250 ml of viral stock, which is plenty - for most proteins optimal
expression with such stock is at 1-10 ml/liter culture for 3 days
(always do optimization though - optimum varies from stock to stock
and protein to protein).
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