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Re: [ccp4bb] protease cleavage sites
 

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CCP4bb <-- 2007 <-- March 2007 <-- 05 March 2007
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Subject: Re: : MTZ to SHELX question
From: "George M {- dot -} Sheldrick" gsheldr {- at -} SHELX {- dot -} UNI-AC {- dot -} GWDG {- dot -} DE
Date: 2007-03-05 		Next message:
Subject: Re: protease cleavage sites
From: artem {- at -} XTALS {- dot -} ORG artem {- at -} XTALS {- dot -} ORG
Date: 2007-03-05


Subject: Re: protease cleavage sites
From: David Briggs bassophile {- at -} GMAIL {- dot -} COM
Date: 2007-03-05
I'd like to third TeV & second 3C ("PreScission").

Both have high specificity, good processivity and I have had a lot of
success with 3C.

I have _never_ got Thrombin to cut cleanly - but I guess I could have been
unlucky...

Dave

On 05/03/07, Cynthia Kinsland wrote:
>
> Hi,
> I'll second the TEV protease suggestion. We use it routinely because it
> is highly specific and easy to make ourselves (and, therefore, cheap). We
> have never seen it cut non-specifically and, since it is cheap, we just
> chuck in a bunch and let it go.
>
> The Prescission protease is also very specific and also available for home
> preparation (it is the 3C protease...prescission is a marketing name). I
> don't have as much experience with it, but it has behaved for me so far and
> I know that a number of people use it routinely with great success.
>
> Another nice thing about having the clones around to make your own is that
> you can make the protease with the same tag that you intend to cut off (say,
> His or GST or whatever your favorite is). Then, you can remove cleaved tag,
> uncleaved fusion protein and the protease all in one post-cleavage step.
>
> In our case, we almost always have a HisTag (often as part of some larger
> fusion) so our TEV is His-tagged. We have some of the GE pGEX vector for
> Prescission protease, so our 3C clone has GST on it.
>
> Best of luck,
>
> Cynthia
>
> On Mar 2, 2007, at 5:01 AM, Rene Frank wrote:
>
> Hi,
>
> A non-ccp4 Q. Sorry.
>
> I would like to use a cleavable purification tag at the
> N-terminus/extracellular end of my membrane protein for purification. Before
> I start, I wonder if someone could recommend a particular protease site that
> I can engineer between the tag and my protein? How about a proprietary
> cleavage system such as the PreScission protease (GE Healthcare)? I would
> be grateful to hear success and horror stories in this area.
>
> Best wishes,
>
> Rene
>
> ================================================
> Dr R.A.W. Frank, PhD
> Royal Commission for the Exhibition of 1851 Research Fellow
>
> Prof Seth Grant Lab / Genes to Cognition
> Wellcome Trust Sanger Institute
> Hinxton
> Cambridge CB10 1SA
>
> Work Tel: 0044 (0)1223 834244 ext. 7318
> Cell No.: 0044 (0)7870 208280
> ===============================================
>
>
>
>
> ____________________
> Cynthia Kinsland, Ph.D.
> Cornell University
> Protein Facility Director
> 607-255-8844
>
>
>
>


--
---------------------------------------
David Briggs, PhD.
Father & Crystallographer
www.dbriggs.talktalk.net
iChat AIM ID: DBassophile
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