Hi John, Thank you very much for all the valuable suggestions and the helpful link. I think it's worth trying S.cerevisiae both for expression and also protecting the environment from that strong Pichia odour.
Krish On Fri, Jul 12, 2013 at 3:17 PM, John Lee <kw...@msg.ucsf.edu> wrote: > 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 >> >> >> >> > >
