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Design and Construction of Novel Intein Mediated Biosensor in Order to Detect Amyloid Fibrils

Miri, Mohammad | 2016

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 49636 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. The most important factor in protein's function and its 3D structure is folding. Misfolding or native folding perturbation in some peptides and proteins force them get into amyloid fibril aggregation. It has been observed that these aggregations are the cause of some debilitative diseases especially ones that are related to aging like Alzheimer disease, Hantington's disease, typeII diabets etc. Recently it has been shown that specific regions in human catalase protein interact with some amyloid fibrils, such as Aß, IAPP, PrP etc. Inteins are self-splicing protein enzymes, which excise themselves from the mature protein and join the two flanking adjacent sequences by a new peptide bond in a post translational modification manner. Recently، inteins have shown widespread applications in protein engineering and development of modern protein based biosensors to detect different interactions of macromolecules with each other and with small molecules. Introduction of a simple and efficient method to detect amyloid aggregations has been always the concern of many scientists in chemistry, medicine etc. In the present study, we aimed to design and synthesize different novel protein biosensors to detect amyloid fibrils in which inteins act as a transducer and different fibril binding sequences as a recognition element.Our strategy has been to insert different motifs of catalase's amyloid binding region into mycobacterium's intein (Mtu-RecA intein). Afterwards, production and purification of these recombinant proteins were done. Finally the new properties of these enzymatic molecular constructs in different situations were investigated.Two recombinant molecular constructs (SMM1 & SMM2), were produced and purified by Ni affinity chromatograph. SMM1 contains only 18 amino acids fibril binding domain and SMM2 contains the entire 29 amino acids of catalase's exon10 as a binding domain. The role of pH, ionic strength, temperature, and amyloid fibrils in intein's splicing was examined. It was determined that at low pH (2-4) SMM1 construct splices itself. SMM1 showed binding to lysozyme fibrils but it was not enough to induce splicing. The initial expression of SMM2 showed a truncated construct that needs further modification and investigation
  9. Keywords:
  10. Biosensor ; Protein Fibrillation ; Amyloid Fibrils ; Intein ; Protein Biosensor ; Protein Splicing

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