Dear Wenhe,
Are there other ligands known for this protein ? Could these be
chemically modified and still interact with your protein ?
Then you could use ligand affinity chromatography to purify your protein
eluting with the same or other ligands.
For me this the only way to obtain sepiapterin with defined co-factor
(cybacron blue column) and/or ligand (sulfapyridine column) occupations.
Alternatively soaking to exchange ligands in the crystal ?
Best of luck,
Ruud
On 21/8/17 05:41, WENHE ZHONG wrote:
Dear CCP4BB members,
We would like to identify a ligand that is present in crystal structure
(according to strong positive densities at active site) but absent in
crystallization condition. We already have some candidates in mind based on our
knowledges on this protein but we need to validate further. The general method
we are using now is to use methanal to precipitate protein and extract ligand
from the precipitated protein. Then we analyse the methanol extraction sample
on LC-MS. One problem of this method is that the methanol extraction will not
be 100% efficient which means there is only a small portion of bound-ligand can
be extracted from the protein— particularly if the ligand binds very tightly to
the protein. So I would like to know whether anyone has experience to
efficiently extract tighly-bound ligands from protein for downstream analysis.
One method is to digest protein with protease such as trypsin. Or use urea to
denature the protein. However, these methods require relatively long processing
time which is not optimal when the ligand that we want to analyse is unstable
(degrade overtime). Anyone has more suggestions?
Thank you!
Kind regards,
Wenhe
--
Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch
