baculovirus expression kits

[ashendel at aclcb.purdue.edu]

On 1 Jun 1994 13:33:02 GMT, 
Sean David Moore  <smoore at mail.sas.upenn.edu> wrote:

>tjames at eagle.wesleyan.edu wrote:
>: I need your help in buying a baculovirus expression kit. If you have experience
>: with a particular kit or have heard good or bad reports on such kits, I would
>: very much appreciate hearing from you.

>I have used Buculo-gold and found it to work well for the expression of
>p53 and Large-T antigen.
>There was an instance when NONE of the batch gave recombinants, we thought
>it was our error and continued on...about two weeks later, we were called
>by the company, they apparently had a bad batch that went out...

Interesting...
I just compared (for the first time) some Baculogold to my do-it-yourself 
linear BakPak 6 (originally from CloneTech) and found the Baculogold 
failed  completely while the BP6 worked.  Maybe it was a bad batch...

As for the original question, I am not a "kit" person, but I did use the 
Invitrogen kit to learn the technique.  It still would  be a good 
learning kit, except it uses the "first generation" technology, and I now 
use the "second generation" BP6 system (CloneTech).  However, I have not 
seen their kit instructions although a neighboring lab has used it 
successfully.  The Baculogold system (Pharmingen) is slightly better 
technically, is somewhat better established and is apparently more 
popular.  However it is considerably more expensive with the list price for 
linear viral DNA of $297 for 0.5 ug, which is enough for 5 transfections. 
The viral DNA must be purchased since the virus is a defective mutant and 
cannot be propagated in normal Sf9 cells.   The BP6 virus is $50 to 80 and 
can be used to make you own linear DNA (this is relatively easy but 
requires Bsu36I).  Baculogold is technically better because it relies on a 
mutation which affects 100% of the virus DNA molecules, even if not 
linearized.  The BP6 system depends (for background reduction) on complete 
digestion of the viral DNA with Bsu36I.  Since digestions are never 
complete, there is always some background with the BP6 system.  However, in 
my hands, the background with BP6 usually is 3% of the primary virus but 
once was as high as 20%.

BTW, the publication describing the basic (first generation) 
baculovirus method and the BP6 development are freely available, the medium 
is sold by Sigma, GIBCO, and most other supply houses, and Sf9 cells are 
available though the ATCC.  A kit is not necessary, nor will it guarantee 
high protein yields.

As for learning this method, the key is to have very high cell viability.  
This was emphasized in the Invitrogen manual and in the Summers and Smith 
(classic) AES publication, but it ***cannot be overemphasized***.  Having 
taught several beginners this method, this is the hardest part to get 
right, even for people that have experience with animal cell culture. 

Despite its shortcomings, I am very enthusiastic about this technology, 
and wish you luck with it.
Curt Ashendel
Purdue University
West Lafayette, IN
ashendel at aclcb.purdue.edu