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
