This project has been running at the Boys Langton for the past eight years. It has been entirely funded by the Wellcome Trust and was the project that launched Authentic Biology, a national collaboration of seven state secondary schools each carrying out their own unique research programme.
The myelin sheath is the protective layer of tissue which is wrapped around the nerve fibre, or axon, in mammalian central nervous systems (the brain and spinal cord). This layer is laid down by cells called oligodendrocytes and is mostly composed of fatty material, although there are several proteins which are important for maintaining the integrity of the sheath. The second most abundant protein is Myelin Basic Protein and this is the protein we have chosen to study.
Myelin Basic Protein (MBP) is a fairly small protein, approximately 20kDa and around 170 amino acids long. It is unusual in that it has no fixed tertiary structure (there are no cysteine residues) so its secondary structure is only held together by hydrogen bonds. It is also subject to a whole range of post-translational modifications ranging from phosphorylation to citrullination, acylation and de-amidation. For this reason, no one has been able to crystallise the protein to allow us an opportunity to determine its function in the myelin sheath.
Our project has involved cloning the human gene for MBP and working with it in Escherichia coli and Saccharomyces cerevisiae and we had to obtain a licence from the Health and Safety Executive to allow us to carry out this transgenic work on a human gene. We cloned the gene into a shuttle vector, a plasmid (or ring of double-stranded DNA) which is capable of “working” in both E. coli and yeast. We positioned the gene behind a Gal-promoter which would then allow us to transcribe the gene in yeast and see if the protein was expressed, we also included a His-tag to help purify the protein.
We were able to demonstrate expression of the human protein in yeast and then used the His-tag to purify the protein by nickel-sepharose chromatography. We then confirmed that it was MBP by western blotting of the purified protein.
Next, we were able to show that the human MBP had been phosphorylated by yeast kinases after it had been translated. We then used site-directed mutagenesis to mutate possible phosphorylation sites and were able to prove that the MBP was being phosphorylated at Threonine-98.
We are currently cloning a second human protein, Proteolipid Protein (PLP1), with a view to developing a protein binding assay to see if various mutations of MBP affect its ability to bind to PLP1. To date, over 600 students have been involved in MBP.