mbp2

mbp squared

Multiple Sclerosis is a neurological disease affecting around 100,000 people in the UK. The myelin sheath, which insulates nerve fibres, gets broken down – this means that nerve impulses can’t be transmitted.

One of the major proteins in the myelin sheath is Myelin Basic Protein (MBP). It is an unusual protein, it lacks a defined 3-D structure and so it can “fall apart” quite easily. If this happens, then the myelin sheath is attacked by an auto-immune response. One possibility is that phosphorylation of the MBP (adding a phosphate group to specific amino acids) triggers the unfolding of the protein.

The project has had a dramatic impact on the numbers of students studying biology in the school. We now have 150 students at AS and 50 at A2, of these 170 are involved in the project, we anticipate that these numbers will be even higher in the future.

We will be looking at how the human MBP protein behaves in the yeast Saccharomyces cerevisiae. This is normal brewer’s and baker’s yeast, but its’ biochemistry is startlingly similar to humans. We will see how the protein is “processed” by the yeast and try to identify which pathways are involved. This will be possible in yeast since there are entire libraries of strains each of which has one single non-essential gene knocked-out and this represents an enormously powerful tool in dissecting the biochemistry involved.

Current state of play on MBP2

We have cloned the human gene for Myelin Basic Protein and added a run of eight Histidine residues to the C-terminus of the protein. We have inserted the gene into a yeast expression vector behind a galactose promoter – this means that we can “switch on” the gene by switching the yeast from glucose- containing media to media with galactose.
We have shown expression of human MBP in yeast using specific MBP anti-bodies and we have purified the protein on nickel-sepharose columns using the His-tail. We have used anti-bodies to try to determine which post-translational modifications have occurred on the protein that has been expressed in yeast.

So far we have identified phosphorylation of the protein on Threonine-98. Analysis by mass spectroscopy has confirmed that the intact protein does carry an additional phosphate group, current spectroscopic analysis is aimed at trying to identify which fragment of the protein carries the phosphate (hopefully the fragment containing Threonine-98). One of the post-graduate students at the University of Kent has used site-directed-mutagenesis to change Threonine-98 into an Alanine residue for us, and we will be transforming this plasmid into yeast to see if we can demonstrate the lack of phosphorylation of this variant of the protein. If this work is successful, we aim to publish a paper in PLOS-one, the manuscript is currently being prepared.

Five years ago, David Colthurst made an application to the Wellcome Trust for a People Award. He had the idea that a novel research programme into multiple sclerosis could be establsihed in a secondary school. The Wellcome Trust agreed to fund this venture and the Myelin Basic Protein Project (MBP2) was born.

2011 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.