The important bit about Thrombin is to find the right source of it. Some
suppliers are *much* better than others. I've had no problems with this
protease in the recent year, but had trouble earlier.
Note that Thrombin leaves 2 amino acids whereas TVMV, TEV, and 3C leave
one amino acid (or even none if you have the right N-terminus).
Artem
> 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 <[EMAIL PROTECTED]> 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
> ---------------------------------------
> Anyone who is capable of getting themselves made President should on no
> account be allowed to do the job. - Douglas Adams
>
