Channel protein reconstitution in lipid bilayer

Irit Rappley via methods%40net.bio.net (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  
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 <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