a comment to add about phosphatase (PP2A, C class serine theronine)
buying phosphatase for crystallization trials was kind of not
practical( very high prices )
so we tried expressing and purify it from overexpressed in bacterium
with yields very low.
Also the purified phosphtase co-purify some prot
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Debreczeni, Judit
Sent: Monday, January 30, 2012 3:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Kinase crystallization
Does your kinase autophosphorylate by any chance? -- That can produce
differently
Does your kinase autophosphorylate by any chance? -- That can produce
differently phosphorylated species and affect crystallisability. You can
detect it by e.g. mass spec, and tackle it by dephosphorylating the
protein prior to crystallisation or by coexpression with a phosphatase.
From: CCP
Staurosporine come to mind as a general kinase inhibitor. I also second
Artem's suggestion that ligands make a big difference, we had several
cases of kinases which required ligands for crystallization success.
Also make sure you eliminate any floppy ends which may interfere with
packing.
Good
It is a fairly common issue with kinases. Among other options you may want
to try a generic kinase inhibitor (there are several good ones just look at
pdb structures for ieas) and if this does not help then you could attempt
to clamp the motion down via an inter-lobe engineered disulphide bond...
