Dear Christine,
Slow crystallization may also be due to some residual protease activity,
which cuts away a flexible loop. You could check this with mass spec or
running a gel with protein from a crystal. If it turns out that you need
proteolysis to get crystals, you could try in situ proteolyis by adding
a trace amount of protease e.g. Trypsin of Chymotrypsin to your
crystallization trials. The link below gives a recent paper in this
subject.
Good luck!
Herman
http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0005094
________________________________
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On
Behalf Of Harman, Christine
Sent: Tuesday, August 14, 2012 7:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Optimizing xtals conditions
Hi all,
I need some advice on reproducing crystals that emerged in about
2 months from a screen. The background is I set up a 96-well hangdrop
screen and checked the tray after 1 week and then every 2-3day for about
3 weeks. I did notice that this particular drop seemed very dynamic
overtime. I stopped looking at the tray for about 2 weeks and then when
I re-checked after that time I found ~5 beautiful single growing
crystals. These crystals continued to grow with a few more emerging
over the next week. I am not sure of the exact time the first crystals
emerged, but between the time I set up the screen to when I saw the
crystals it was not quite 2 months (~1 week short). I am in the process
of reproducing this hit. I set up drops with the protein at the same
concentration used in the screen (~5mg/mL) and at a higher concentration
~2.5X. This protein is a Fab that was complexed with peptide before
setting up initial screen. The protein is in only 0.1M Tris pH8.0. The
crystal condition is 13.4% PEG 8K, 2% MPD and 0.1M imidazole pH6.5. I
have checked drops (~3 weeks old) and with the more concentrated protein
I see some interesting crystalline like ppt similar to what I see in the
drop in the screen; however, no crystals yet. The drops with the lower
concentration of protein are still somewhat clear with some having
crystalline like ppt. I varied the PEG 8K concentration 12.5-14% vs MPD
at 2, 4, and 6%. What I need advice on is what else can I do to speed up
the crystallization process. Does anyone have suggestions besides
increasing the protein concentration. I have limited amounts of protein
left for optimization so I was wondering if there were any additives
that were better known to facilitate faster crystallization as opposed
to testing everything under the sun. Any suggestions would be great :)
Thanks,
Christine
