Expression and Purification of Amyloid Beta Recombinant Peptide and the Effecs of Ultrasmall Peptides Enriched in Branch Chain Amino Acids as Inhibitors in Amyloid Formation
Rezaei Adariani, Soheila | 2014
- Type of Document: M.Sc. Thesis
- Language: Farsi
- Document No: 46225 (03)
- University: Sharif University of Technology
- Department: Chemistry
- Advisor(s): Kalhor, Hamid Reza
- Nowdays, it has been shown that protein misfolding is involved in more than forty diseases including Alzheimer’s and Parkinson’s. Alzheimer’s disease is one of the most common neurodegenerative diseases that disrupts brain function. The main cause of this illness is due to formation of extracellular amyloid plaque from a 42-amino acid-peptide called amyloid beta. Amyloidosis is caused by missfolding of peptides and proteins mainly because of physical and chemical changes in biomolecule or their environment.
Therefore, in this dissertation, using synthetic and recombinant abeta peptide, kinetic of amyloidosis was examined. Ultrasmall peptides (three-amino acid), containing branched chain amino acid, were designed in such a way that most permutations were considerd; their roles in inhibiting amyloid formation was investigated.
In realizing that small amount of synthetic amyloid peptide is costly and uneasily accessible, we decided to express and purify recombinant abeta peptide. To meet this goal, amyloid beta gene (via a bacterial DNA plasmid) was expressed, and subsequently using anion exchange chromatography the amyloid beta peptide was purified. Furthermore, using tandem mass spectroscopy the peptide sequence was verified.
Moreover, due to the involvement of large proteins in misfolding diseases, Hen Egg White Lysozyme (HEWL) was investigated in high temperature and various pH, as a paradigm for studying amyloid formation.
Since amyloidogenic region of abeta peptide has been found to be located at position 16 to 22 (lysine- leucine-valine- phenylalanine- phenylalanine-alanine- glutamatic), six novel peptides, which included leucine, isoleucine, valine (KLI, KLL, KVI, KVL, KVV, KII), containing beta hydroxyl amino acids resembling amyloidogenic region of amyloid beta,were designed as inhibitors. The positive charge of the peptides (due to lysine), can bring about a better solubility and interaction to which mimic N-terminal of amyloidogenic regin of the target (abeta peptide).
Furthermore, in the present dissertation, self-assembly of the ultrasmall peptides enriched in branched chain amino acids was explored, in a number of conditions such as various pH, and different ions. Self-assembly of peptides has been known for fibril formation. All in all, the roles of KVV and KVL peptides were studied as inhibitors for amyloid beta formation. Results show that these two kinds of peptides act specially as inhibitor of amyloid fibril formation, while they do not affect HEWL amyloid formation
- Peptide Amyloid Beta ; Amyloid Inhibitors ; Ultrasmall Peptides ; Branch Amino Acids