1) Yes. They work "OK" - but I prefer to use dedicated vectors mostly for
aesthetic reasons, since the TriEx series are kind of bloated (for a good
reason). The expression levels are usually alright. So, in conclusion - if
you're trying 100 genes in all kinds of systems you could use TriEx (or
Gateway, LIC, etc.) but if you have 10 genes to try I would recommend
regular vectors.
2) bac-2-bac is a proven, fairly robust system that incorporates an
integrity check at an early stage (bacteria). Also you can sequence the
prepared bacmid to verify that everything is OK. With the other two systems
you're saving a step but (in my opinion) you're reducing your confidence
level. With the Gus indicator there's a degree of control but it's a PITA to
stain and verify, etc. Direct transfection of pure bacmid is easy, and you
get first-passage virus that's almost always good enough to infect massive
amounts of cells, so your overall time savings are (in my opinion) greater.
3) STREP tag vs His tag - kind of like comparing a sports car to a lorry.
Sometimes you win using one but not the other - however for a generic case I
would nearly always prefer the truck. STREP can be essentially one-step, but
the yields are often quite poor due to low resin capacity and fragility; the
resin is expensive and does not always regenerate well. In comparison,
His-tag nearly always works, usually gives loads of protein (as much as your
system can express in a competent form), gives adequate purity (if you use
His-Select or Talon in combination with detergent lysis you get only a
couple of foreign bands) and is cheap to use and easy to regenerate. So, if
you're working with something crazy or esoteric - some 250 kDa protein that
only expresses in the cerebrospinal fluid of recombinant Madagaskar Chipmunk
embryos - then STREP tag is probably better. For an 'easy' protein expressed
in bacteria, baculovirus, or yeast - His tag is a reasonable default
starting point.
Artem
-----Original Message-----
From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
Anirban Adhikari
Sent: Thursday, December 20, 2007 5:56 PM
To: [log in to unmask]
Subject: [ccp4bb] Question about protein expression (not CCP4 related)
Hello,
Sorry about the non CCP4 related question. I have few questions regarding
protein expression:
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!
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?
3. Is anyone purifying their protein using the Strep-tag II/Strepactin
affinity system and if so how does it compare to His-tag?
I am looking forward to reading your opinions and suggestions.
Thanks a lot,
Anirban
Anirban Adhikari, Ph. D.
Postdoctoral Research Scholar
Department of Molecular Biology
UT Southwestern Medical Center
5323 Harry Hines Blvd,
Dallas, TX 75390-9063
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