Channel protein reconstitution in lipid bilayer

Fulvio Celsi via methods%40net.bio.net (by fulvio.celsi from gmail.com)
Fri Jan 21 04:02:00 EST 2011


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 <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 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 <mailman.743.1295455841.15153.methods from net.bio.net>, Fulvio
>> 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/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
>>>
>> _______________________________________________
>> Methods mailing list
>> Methods from net.bio.net
>> http://www.bio.net/biomail/listinfo/methods
>
> _______________________________________________
> Methods mailing list
> Methods from net.bio.net
> http://www.bio.net/biomail/listinfo/methods
>



More information about the Methods mailing list