Hi Jenny, there are probably as many 'folding enhancers' as there are crystallization additives. Arginine might only be the most popular, followed by things like proline and other amino acids. The mode of action of those can partly be explained by their mild chaotropic properties. For a difficult refolding project, the conditions should be screened sytematically. Other things to try would be sucrose, glycerol, PEG in the range of several % etc. with purified chaperones at the end of the row. For initial experiments, I would always go for the quick dilution method! Dialysis is popular in many protocols, however a lot of them were initially developped in / for an industrial context where it makes a big difference if you have a procedure on a 1kg scale needing a 1000 l or a 1000 m3 vessel. In the lab you don't care if you need 10 ul or 10 ml of refoding volume. Other sometimes successful methods are refolding on a column e. g. Ni-NTA, if you have a his-tag, if not, try a gel filtration column. In case you expect disulphide bridges you will have to take care about them during the development of your protocol by adjusting the redox potential (possibly in a stepwise manner).
Good luck! Clemens Quoting Jenny <[EMAIL PROTECTED]>:
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
