what chemicals can be added during purification to inhibit protein protein interaction?
We have that using the MOCR domain as a solubility tag has greatly aided in the purification of several poorly folded and aggregation prone proteins.
http://www.ncbi.nlm.nih.gov/pubmed/18824232
Hi Sunil,
You could try low concentrations of detergents like Tween-20 or SDS?
Good luck!
Hi Sunil,
as always, the means to suppress aggregation crucially depend on the properties of your protein. Adding salt so prevent protein/protein-interaction is usually a good idea ("salting-in-effect"). Too much salt however will again "salt-out" the protein, by preferred interaction of water with the salt ions.
I have experienced good results by adding arginine or glutamate in mM concentrations.
Trehalose, betain, ectoin, sucrose, sorbitol and other sugar-alcohols are used to prevent aggregation in concentrations up to 4 M. Which best solubilizes your special protein must be determined empirically, I'm afraid.
Have a look into this paper:
http://aem.asm.org/content/77/13/4603.full
There is also the new (and to mind, rather plausible) hypothesis that proteins more likely aggregate during cell lysis, not during expression itself:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052482
Best of luck!
Hi Peter,
Thank you for your insightful suggestions. I am using l-arginine during cell lysis and i am observing a substantial improvement in protein solubility. However, protein aggregation still remains same.
Thanks
Sunil Singh
Try to empirically optimize the pH and salt content of your buffer. There should be a combination your protein is happy in.
Especially pay attention to the isoelectric point of your protein. Choose the pH of your buffer at least 1 pH-unit off the pI (in either direction), otherwise your protein will lose net-charge and aggregate/precipitate.
Some proteins aggregate unless they are denatured during purification. In those cases, you'll probably need to include 6-8 M denaturant in your buffer--i.e., urea or guanidine hydrochloride. This can be desalted out just prior to experiments that require the protein to fold in its natural state.
We have that using the MOCR domain as a solubility tag has greatly aided in the purification of several poorly folded and aggregation prone proteins.
http://www.ncbi.nlm.nih.gov/pubmed/18824232
Hi Jeffrey,
Thanks a lot for suggesting a better solubility tag for recombinant protein production.
Thanks & Regards,
Sunil Singh
Hello Dear Sunil,
u can try denaturing purification. I would like to suggest first use Urea denaturing purification. Various protocols are available if u dont find let me know i will suggest some literature.
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