Results

Following the successful transformation of yeast with the pSLB1 plasmid, we have been able to show expression of Human MBP in yeast.  We used a host strain with a Ura3 mutation, this means it can only grow when Uracil is included in the growth medium.  However, once the yeast is transformed with the pSLB1 plasmid, the yeast is now able to grow in Ura- media, since the Ura3 gene on the plasmid compensates for the mutation.
The MBP gene is spliced into the plasmid just behind a Gal promoter, if we switch the yeast from glucose-containing media to media with galactose in, then the promoter starts to transcribe everything downstream of the promoter, in this case, human MBP.
We prepared a totla cell lysate from these cells by boiling them with sodium hydroxide and then with SDS-sample buffer.  We ran identical gels, one was stained, the other was blotted onto nitrocellulose membrane and probed with anti-MBP antibody.
The purified MBP that we bought as a positve control for the blots shows up clearly, the three tracks of yeast lysate, 5, 10 and 15 ul, show an increasingly strong signal for human MBP.  The next step is to purify the MBP using nickel affinity chromatography and then look for phosphorylation of the MBP using phopho-amino acid specific antibodies in the western blots.

SDS gel of expressed MBP
Western blot of expressed protein