Channel protein reconstitution in lipid bilayer

Irit Rappley via methods%40net.bio.net (by irappley from scripps.edu)
Thu Jan 20 12:54:39 EST 2011


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
>>
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