Hi Krish, Seconding Tim's comments, you may need to switch construct or expression host. I've seen many times when a membrane protein seems to be in the yeast membrane, but seems unextractable or most likely, improperly folded. Since you're already working with yeast, I would recommend trying S.cerevisiae. It's worked for many integral membrane proteins, and smells a whole lot better than Pichia.
http://csmp.ucsf.edu/pdf/Pub14_PMID20946832.pdf My 2 cents. Good luck, John On Fri, Jul 12, 2013 at 1:34 PM, Timothy Craig <tlmcra...@hotmail.com>wrote: > Hi Krish, > You may need to do some construct engineering to get your protein to > express well to the plasma membrane. These steps involve creating many > expression construct variants including N and C-terminal truncations, > fusion partners at the N-or C-termini, or even signal peptides at the > N-terminus to properly target your protein to the plasma membrane. Pay > attention to post-translational modifications as well. You may also want > to look into protein trafficking signals, which can be found in a nice > paper by Duvernay et al 2006, which is focussed on GPCRs but applies to > other membrane proteins as well. Additionally, you may want to try other > expression systems like BV, mammalian, or even e.coli with all of these > variants. Of course, if you just want to try more detergents you can > purchase a detergent screen from (sorry for the self-plug) Emerald Bio to > test a larger amount of detergents than you probably have thus far. > Expressing membrane proteins can be a daunting task, but with enough hard > work you can be successful. > > Best of luck > -Tim Craig > Emerald Bio > > ------------------------------ > Date: Fri, 12 Jul 2013 00:29:28 -0400 > From: krishna....@gmail.com > Subject: [ccp4bb] Membrane Protein expression and purification > To: CCP4BB@JISCMAIL.AC.UK > > > Dear All, > > First of all sorry for bringing the non-ccp4 post. As our CCP4 community > is filled with experts in various fields of structural biology: I'd like to > get some help/suggestions from the membrane proteins expert community. > > I'm trying to express my membrane protein in Pichia pastoris (SMD1163H). I > see my protein getting expressed but the yield is very low. I tried > optimizing the media, temperature and additive like DMSO but none of them > improved the yield. Apart from that, the main problem is my protein is not > completely extractable from the Pichia membranes. Tried DM, DDM (2 to 4%), > beta-OG, LDAO,CHAPS and C12E8 (2 hrs to over night in 4 C and 1hr at RT). > Only I could get little bit of my protein in DDM. If I spin my protein even > at 30K to 50K g after extraction most of it is ending up in the pellet. I > know every protein behaves different but I'd like to know - Did anyone > observe the same behavior with Pichia membrane protein expression? I'm even > trying with the interaction partner (chaperone like) to express it in happy > form! > > I'd like to get some suggestions regarding the expression in Pichia or any > other system that might help. Any tips on the solubility part of membranes > would be really helpful. > > > Thank you. > > Krishna > > > >
