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
e-mail: [EMAIL PROTECTED]
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