Hi John,
This is really an amazing wild west story: the man who crystallizes
faster than his protease! I really must compliment you with how you
successfully performed these experiments!
Unfortunately, proteins usually do not crystallize that fast (at least
not in my hands), so in these cases other methods have to be used. As
has mentioned before, protease inhibitors are the way to go. Especially
with autolysis, as one protease cuts another one, the speed of the
reaction goes with the square of the protease concentration. Whereas in
dilute solutions not much happens, as soon as you start to concentrate
towards crystallization conditions, say 10 mg/ml, degradation suddenly
goes very fast.
There are 2 cases to consider:
1) the protein you want to crystallize is a protease and is destroying
itself. In this case you need to cocrystallize with a potent and
specific inhibitor. With serine proteases, Wolfram Bode was very
successful by using chloromethylketone-containing peptides (e.g. PPACK).
These compounds would make covalent links with both the active site
serine and histidine, effectively killing any protease activity.
2) the protein you want to crystallize is not a protease and it is a
contaminant which is causing the problems. In this case I would add a
protease inhibitor coctail in an earlier step of the purification to
block the protease before the final purification steps. I would also add
some small broad protease inhibitor e.g. PMSF to the protein solution
used for crystallization.
Herman
________________________________
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of John Domsic
Sent: Wednesday, January 16, 2013 2:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein degradation in crystal
Hi Lisa,
Speed is definitely a big factor here. With a protein I work
with I can get large crystals in myriad conditions that only diffract to
about 4-5 Ang. What I ended up doing was taking these crystals and
seeding entire screens. I found that not only would crystals appear
sooner but it revealed novel crystallization conditions. These seeded
crystals would appear within minutes as Preben described and diffracted
to better than 2 Ang. Another thought would be to try limited
proteolysis to see if you can identify a more stable construct.
-John
--
John Domsic
Postdoctoral Fellow
Gene Expression and Regulation Program
The Wistar Institute
Philadelphia, PA 19104
On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth
<j.p.mo...@ncmm.uio.no> wrote:
Dear Lisa
It is not uncommon to see breakdown products when you
run crystals on gel. Espesially if they are older crystals, sometimes
you even see higher molecular bands, these are probably due to intra
molecular cross links formed over time.
If you are worried about stability, try to increase the
crystallization speed, we have one example where we see a clear
difference in both crystal quality and even space group depending on
when we fish the crystals. The crystals appear within 5 min, the best
quality data sets come from crystals we fish after only 30-60 min.
You may also have a little protease contamination of
course, to prevent this add protease inhibitor, or DTT, or EDTA to you
protein before you set it up.
cheers Preben
On 1/16/13 12:14 PM, LISA wrote:
Hi All,
I have an 36KD protein which can be crystallize
in two days. Most of the crystals are very big. But all cystals have
poor resolution,lower than 3.8 A. I picked some crystals, washed them in
the mother solution and then run SDS-PAGE. It is surprised to find that
different cystals have different components. Some crystals have several
samll bands below the band of the protein. And in some crysals the
bigger size band (as the construct should be) almost disappared and have
smear. Does the protein was degradated in the crystals? Did someone met
the similar problem as I? Thanks
All the best
lisa
--
J. Preben Morth, Ph.D
Group Leader
Membrane Transport Group
Nordic EMBL Partnership
Centre for Molecular Medicine Norway (NCMM)
University of Oslo
P.O.Box 1137 Blindern
0318 Oslo, Norway
Email: j.p.mo...@ncmm.uio.no
Tel: +47 2284 0794 <tel:%2B47%202284%200794>
http://www.jpmorth.dk
