??Help: Best Endoprotease ??

Marianne Ostermayer ostermayerm at ubaclu.unibas.ch
Wed Aug 26 03:01:41 EST 1998


Well, my guess is that you will not be happy with my answer, but if you
dont want to use one of the vectors around...
-find out in which cleavage buffer your protein is soluble, then use
that one
-you can introduce a methionin and do chemical cleavage
-choose the cheapest endoprotease
-choose the one that is the most stable
-choose the one that is easy to get rid off afterwards
-choose to engineer a his tag instead
-think about what you use your protein for afterwards, some fusionparts
can cause trouble in downstream applications

The consensus (if there is any) is: let's have a couple of cups of
coffee, think, have another cup of coffee, make a decision, argue about
it, then go for it.

Alternatively, you can do a literature search on recombinant proteins
and
-find out what is the most popular system
-find out the fraction of cleaved protein
-find out the most popular protease
-post those results here
-make up your mind what to use

The least scientific way to decide is to buy the protease from the (most
pretty/handsome) (any criteria can be inserted here) sales
representative. Of course, this is no guarantee that it will work in
your construct, but there is no guarantee that it does not work in your
construct either.

Marianne Ostermayer
Swiss Tropical Institute
Socinstrasse 57
4002 Basel

S. Findley wrote:

> I need to engineer a protease cleavage site into a construct.
> Thrombin? Xa? Prescission? TEV?
> Is there a consensus out there?
>
> Thanks.
> SDF
> --
> Seth D. Findley               polliwog at u.washington.edu
> Department of Biochemistry    Phone: 206-543-1710
> Health Sciences Building J579
> University of Washington, Box 357350, Seattle, WA 98195






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