Channel protein reconstitution in lipid bilayer
(by irappley from scripps.edu)
Fri Jan 21 12:01:58 EST 2011
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
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.
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
> 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 <irappley from scripps.edu>:
>> 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
>> 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
>> 3. Having lysed your cells with the optimized detergent protocol,
>> IP your
>> protein or protein complex. Again, lots of troubleshooting will be
>> before this works well.
>> 4. Prepare a film from the lipids you want to use (see
>> 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
>> 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
>> amount of detergent might still leave enough protein activity for
>> Hope this helps.
>> On Jan 19, 2011, at 6:18 PM, DK wrote:
>>> In article <mailman.743.1295455841.15153.methods from net.bio.net>,
>>> Celsi <fulvio.celsi from gmail.com> 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/
>>> 4. Mix with lipids and slowly dialyze out (or remove by some other
>>> means) the detergent. Depending on conditions/lipid mixture,
>>> of various sizes can form. Giant liposomes can be used in patch
>>> 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.
>>>> Fulvio Celsi
>>>> Sector of Neurobiology,
>>>> International School for Advanced Studies,
>>>> Scuola Internazionale di Studi Superiori Avanzati (SISSA),
>>>> Via Bonomea 265 34136
>>>> Office: +39 0403787 771
>>>> Mobile: +393286489131
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