Abstract
Human saitohin, a newly discovered protein encoded by the intron between exon 9 and 10 of the tau gene, a risk factor for many neurodegenerative diseases, has been recently found from GST -pull-downs to interact with peroxiredoxin 6, a protein with both peroxidase and phospholipase activity. Saitohin has been demonstrated to have allelespecific correlation to tau pathologies by studies, which suggest the interaction of saitohin and tau in a protein pathway. Additional evidence of the interaction of saitohin and peroxiredoxin 6 include the finding that Saitohin 7R allele and peroxiredoxin 6 mutant C47S together increase the splicing of exon 10, misplaced in many neurodegenerative diseases. Moreover, the expression of peroxiredoxin 6 is high in patients with Pick disease, which is related to diseases correlated with the Q allele of saitohin. In order to find out in more detail the mechanism of interaction between saitohin and peroxiredoxin 6 and how it can influence ex on 10 splicing, which in turns leads to neurodegeneration, I've purified and grown crystals from saitohin and peroxiredoxin. In this paper, we report the cloning, expression, purification, and crystal growing of the two proteins, for whose native members no crystal structures are currently available. GST and size column purification yielded peroxiredoxin 6 with a purity of 90%. The results demonstrated the formation of inclusion bodies and high challenge in the refolding of saitohin, which precipitated completely in 10 mM Tris (pH 7.0) and 1mM DTT. The data also indicated the great difficulty in growing crystals from peroxiredoxin 6, which precipitated in most screening conditions. The experimental challenges and accomplishments of this project will help future attempts in the purification and crystal growing of the two proteins.