Hi Jenny,
check on non-reducing SDS PAGE whether your disulfide bond (I assume
there is one) is correct, i.e. you have monomeric protein on SDS-PAGE.
If there are disulfide linked oligomers, try expression in Origami
strain at lower temperature, e.g. 25 deg C.Another possibility is to
direct expression to the periplasm
(http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides#Expression_of_proteins_containing_disulfides)
Good luck!
Guenter
Jenny wrote:
> Dear CCP4 community,
>
> Sorry for this off-topic protein purification problem.I'm trying to
> purify an immunoglobulin-like beta sheet protein with a c-terminus
> HIS construct. The protein expressed both in the supernat and pellet (
> majority ). I purified the supernat and after run gel filtration, it's
> in the void volume. I also tried to purify from the pellet,and do
> dialysis refolding ( with and without L-arg ), after overnight
> dialysis, I run the gel filtration column, the apparent molecular
> weight looks like dimer/trimer. But when I did a CD scan of the
> protein, it's showing an unfolded protein profile.I was wondering if
> there is anyway to promote folding,or if anyway that can make some
> mutations to make it foldable.Any input would be useful.
>
> Thanks a lot.
>
> Jenny
--
***********************************
Priv.Doz.Dr. Guenter Fritz
Fachbereich Biologie
Sektion Naturwissenschaften
Universitaet Konstanz
http://www.biologie.uni-konstanz.de/fritz
Universitaetsstrasse 10
Postfach M665
D-78457 Konstanz
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Tel. Office: +49-(0)7531 88 3205
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