[Protein-analysis] Re: protease
(by hzhen At freeuk.com)
Mon Dec 11 17:26:06 EST 2006
On Mon, 11 Dec 2006 16:20:49 +0100, Abla Sipanis
<ablasipanis At yahoo.co.uk> wrote:
>I'm looking for a protease that has a very specific cleavage site, and
>quite long so that it wouldn't cut at all in the whole Arabidopsis
>proteome. Do you think that this does exist ?
>Amersham for example claims that the recognition site for Prescission is
>LeuGluValLeuPheGln/GlyPro. Does it mean that this is really the only
>sequence that will be cut ? And by the way, I would like to express this
>protease in vivo, so that I would need a cDNA. Perhaps difficult for a
>patented enzyme ?
I very much doubt that it would cut only at that specific sequence.
You can check the literature. Prescission is actually 3C protease
from human rhinovirus, so have a look. If I remember correctly, it
can cleave a number of different sequences.
We make our own 3C protease from HRV 14 although I'm not sure if it is
the exact same one as Prescission (not really sure if there are
different kinds of 3C proteases from HRV, have a look in the
literature). The one we use belongs to another lab, so can't offer it
to you myself, but it's something you can clone it yourself. It is
fused to GST in pGEX and needs to be expressed at low temperature (18
to 20 deg C) or else it would form inclusion body.
>Any suggestion would be ighly appreciated.
>Thanks for you help !
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