From my experience (which is indirect -- I've seen others setting up
projects like this but haven't done it myself) Sf9 cells are probably
the way to go. It's considered not terribly expensive. If you use a
cheap transfection reagent like PEI (polyethylenimine 25kDa, linear,
which you can buy in powder form and dissolve in water at 1mg/mL)
then you really only have the expense of the media and DNA.
A 6-His tag could be very convenient for purification as you point
out. You will definitely need to test for possible interactions with
detergents.
As for time, from what I have seen a project like this could take 1
year just to set up, especially if you don't have experience with any
of these techniques. If you can find someone in a nearby lab who can
help with any part of this project -- even if you have to ask 5
different people, each one for a different aspect of your project --
that would be very helpful. In the end, though, it will just come
down to a lot of trial and error.
Good luck!
Irit
On Jan 21, 2011, at 1:02 AM, Fulvio Celsi wrote:
Thanks DK and Iris for useful suggestions! and for confirming me of
the "not-use" of a kit....
few questions....being a mammalian protein, it will be better to use
Sf9 system? also I'm worried that in the classical E.Coli system this
protein could in inclusion bodies or be toxic..(it's big and lot
hydrophobic). I have no experience with this system (always worked on
E.Coli). it is expensive or difficult to use? or maybe better to use
yeast? (increase yeld..?)
for purification....I have already myc-tagged, but I'm wondering if
maybe it would be better to put a 6-His tag...cheaper than Ab and also
easier...but how much the presence of detergents can influence the
binding of this tag? (I know already for the Myc..and it's quite a
lot..)
Thanks again!! :)
and p.s in your experience...how long a project like this could take?
I was thinking between 6 monts-1 year (if everything goes more ore
less well), but maybe I'm an optimist....
2011/1/20 Irit Rappley <[email protected]>:
I can add a few details to DK's general protocol:
1. Expression in E. coli, Sf9, or mammalian cells -- whichever
works best
for your protein. If your protein is glycosylated or undergoes other
post-translational modifications, you will probably need insect or
mammalian
cells.
2. Detergents can be the hardest part of the assay to
troubleshoot. Try to
find the mildest detergent that works for your application. CHAPS/
CHAPSO is
commonly used for this type of application, but there are many other
options.
3. Having lysed your cells with the optimized detergent protocol,
IP your
protein or protein complex. Again, lots of troubleshooting will be
required
before this works well.
4. Prepare a film from the lipids you want to use (see
http://www.avantilipids.com/index.php?
option=com_content&view=article&id=1384&Itemid=372
for some basic information). Add your IP'ed proteins to the hydration
solution, then sonicate or extrude to form proteoliposomes. From my
experience, detergents cannot be dialyzed out of such a system
because they
"stick" to the lipids that you want to keep. A product called "bio
beads"
can be used to remove the detergent if necessary. For some
applications, a
small amount of detergent is acceptable and might not interfere
with the
function of your protein -- or more precisely, interference from a
small
amount of detergent might still leave enough protein activity for
your
application.
Hope this helps.
Irit
On Jan 19, 2011, at 6:18 PM, DK wrote:
In article <[email protected]>,
Fulvio
Celsi <[email protected]> wrote:
Hello to all
a question for you...so, my PI asked if it is possible and how
much is
difficult to synthetize and produce a transmembrane protein and to
insert it into a lypid bilayer. The biochemist inside me started to
scream..but my background as biochemist is quite old. So..any
suggestion on how it can be done? I saw I kit from Invitrogen
(membraneMax) that state that is quite easy (as every kit in the
world). Anyone has used it??
thanks a lot in advance! :)
Yes it is possible and it has been done many times.
1. Expression as usual (transmembrane proteins are challenging and
yields are almost always low).
2. Find high CMC detergent that preserves protein activity (may take
a lot of screening).
3. Purify (can be difficult but getting much easier with tags/
antibodies)
4. Mix with lipids and slowly dialyze out (or remove by some other
means) the detergent. Depending on conditions/lipid mixture,
liposomes
of various sizes can form. Giant liposomes can be used in patch
clamp
directly, others can be fused with planar bilayers or fused
together to
form larger liposomes.
No kit will ever work for all proteins. I doubt any kit can even
work for
most proteins.
DK
Fulvio Celsi
BsC,PhD
Sector of Neurobiology,
International School for Advanced Studies,
Scuola Internazionale di Studi Superiori Avanzati (SISSA),
Via Bonomea 265 34136
Trieste
Italy
Office: +39 0403787 771
Mobile: +393286489131
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