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 CraigEmerald Bio
Date: Fri, 12 Jul 2013 00:29:28 -0400 From: [email protected] Subject: [ccp4bb] Membrane Protein expression and purification To: [email protected] 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
