There are simple ways for lysis of pichia to prepare cell lysates for SDS-PAGE. One can also use commercially available lysis buffer (see the link below).

Link: http://www.sigmaaldrich.com/life-science/proteomics/recombinant-protein-expression/cell-lysis/yeast-cell-lysis.html

We usually spin down the cells and resuspend the pellet in sufficient amount of 1x Laemmli buffer, boil it and sonicate (only a few seconds, just to get rid of nucleic acids). Then you can load on a gel.. This is just for checking the expression, making cell lysate for purification is a bit different. Sometimes the sonication in PBS is sufficient, but since you have a membrane protein, it can remain in a pellet after sonication and centrifugation. We resuspend the pellet in buffer with 8M urea and some detergent (2% Triton X-100, 0,1% SDS), then mix together with a sonicate and use for isolation

Hi, 

Thank you all for replies.  It seems that the membrane protein I am working on is not as problematic as expected. I did the very basic things and it gave me very clear band. I lysed the cells using sonication in a sonication buffer containing PMSF and DTT.  I did not boil the sample, but I incubated it with SDS loading buffer and BME at 37 degree Celsius. Thats all but there was gel shifting because of incomplete denaturation I guess. I am going to do a western to confirm it. Otherwise, my gel look excellent with minimal background

Once again, thank you all for your suggestions. 

Hi all, 

I did the western and we confirmed it. 

Girish