Loading...

Production and Purification of Recombinant Amylin Peptide and Investigating the Effects of Synthetic and Natural Products on Amyloid Fibril Formation

Sherizadeh, Saied | 2014

706 Viewed
  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 46287 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): KAalhor, Hamid Reza; Matloubi Moghaddam, Firouz
  7. Abstract:
  8. What determines the function of a protein, after its synthesis by the ribosome, is its unique three dimensional structure. The unique structure of protein is achieved through process of folding which is detrimental to protein function. Although this unique structure is stable in a variety of situations, the protein may undergo conformational change, due to slight changes in physiological conditions, affecting the protein structure and function. In certain conditions, the conformational change brings about misfolding of the protein leading to protein aggregation. The protein aggregation can also result in amyloid formation in which a soluble protein is converted to fibrils with specific morphology and dimension. Amyloid formation has been determined to be involved in a number of human disabling diseases such as Alzhimer, due to abeta peptide fibrillation, and in diabetic type II, due to amyloid formation of small peptide known as Amylin.
    In this dissertation, three main goals have been pursued. First, in order to work with an experimental model in studying amyloid formation, the production of recombinant amylin peptide (a 37 amino acid- peptide) in eukaryotic yeast cell have been planned. Amylin, in the patient with diabetes type II has been found to undergo conformational changes, resulting in amyloid formation. In order to produce the recombinant amylin, the gene encoding the amylin peptide was amplified using Polymerase Chain Reaction (PCR) and subsequently subcloned into an appropriate yeast expression vector. Using homologues recombination, the cloned amylin gene was integrated in yeast genome to achieve a stable expression of the recombinant amylin. The expression of the recombinant amylin was examined in the media using SDS-PAGE electrophoreses.
    In the second goal of this dissertation, isolation of natural products from different species of plants including Borage. were carried out, and subsequently their inhibitory effects on amyloid formation were examined. Interesting, the plant. showed strong amyloid inhibitory effect which the responsible molecule turned out to be in alkaloid enriched fraction.
    Thirdly, synthetic approaches were taken to synthesize novel chemical compound based on curcummin. Curcuma, a household additive, has a significant amount of curcummin (1E,6E)-1,7-bis (4-hydroxy- 3-methoxyphenyl) -1,6- heptadiene-3,5-dione) which has been shown to have a number of biological activities including anti-amyloidic. One of the major synthetic approaches have been using diazin derivative as electrophilic compound in reaction with carbanion form of curcumin
  9. Keywords:
  10. DNA ; Electrophoretic ; Amylin ; Amyloid ; Diabetic Type II ; Natural Products ; Vector ; Recombinant Piptide

 Digital Object List

 Bookmark

No TOC