1. I was wondering if anyone used the "Multisystem Expression" vectors from Novagen and if so whats your opinion about these vectors? These are the pTriEx series of vectors that can be used for protein expression in E.coli, baculovirus and mammalian cells using the same construct. Since most of the proteins in our lab is expressed in many different systems the pTriEx series of vectors appear particulary useful!
No experience with those.
2. We are currently using the Bac to Bac system from Invitrogen for generating baculovirus which requires purification of the bacmid DNA from DH10Bac cells before transfection. Looks like the newer systems, such as BaculoGold (BD biosciences) or BacVector (Novagen) systems skip this step altogether and saves time. Does anyone have experience with both systems and if so which one would you prefer?
These are "traditional" systems where you homologous recombination between viral DNA and your transfer vector occurs in insect cells. For these to work sufficiently well, you either must buy cut viral DNA for co-transfections (or prepare it yourself, which is not trivial to do exactly right) or engage in tedious and sometimes time consuming screening of viral clones. Plus, cut viral DNA is *very* expensive. And no matter what, about 10% of the viral clones there would still be WT (which frequently propagate better), so time consuming plaque purification is pretty much required with all traditional system. Bac-to-bac was designed precisely to circumvent this. Of course, it is also not without disadvantages - although initial population of viruses is homogenous, there seems to be some instability with the expression levels (but not viral titers) being reduced with every amplification passage.
If you are (or anyone else) interested, I made modified version of pFastbac that enables cloning artefacts-free insertion of any gene pretty much regardless of the restriction sites present or absent in the insert in any of these variants: 1) no tag, 2) N-terminal TEV-cleavable 8His (6His does not work well enough for insect cells), 3) non-cleavable C-terminal Strep-tag.
3. Is anyone purifying their protein using the Strep-tag II/Strepactin affinity system and if so how does it compare to His-tag?
Strep-tag based purification gives much cleaner protein but the sorbent has lower capacity and is a lot more expensive. One option is to make streptactin-sepharose in the lab (which would be pretty straighforward; about the same as making rTEV protease in the lab).
Dima
