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Molecular Engineering of Recombinant Human Transglutaminase 2 with Correct Folding ( Soluble ) for Studying Promiscuous Organic Reactions

Gholami, Amir Hossein | 2021

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  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 54212 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. Transglutaminases of family enzymes are implicated in different functions in cell including cellular division and cell adhesion. Several members of this family catalyze formation of isopeptide bond that involved in wound restoration and angiogenesis. Transglutaminase 2 that have been studied in this project, are found in almost every human cell tissue. This enzyme has multiple functions depending on its cell location. In cytoplasm TG2 act as GTPase and is involve in post translational modifications. This enzyme by using calcium form a isopeptide bond between two proteins, isopeptide bond is function in brain cortex and cells creates amyloid structures that including brain diseases, Parkinson and Alzheimer diseases. Recombinant expression of this enzyme in bacterial cells is associated the formation of inactive protein masses (aggregation), which precludes in vivo studies on the activity and structure of this enzyme.In this project we attempt to make changes in structure of this enzyme and using structural engineering to receive expressed soluble protein. our plane has been to create soluble protein, this approach noun as directed evolution by creating random mutations in the gene sequence of this enzyme. A recombinant molecular structure was synthesized in the form of a circular DNA that provides the ability to distinguish soluble from insoluble TG2 proteins at the surface of bacterial colonies. In the design of this structure, fusion of two proteins using a peptide interface was exploited and implemented by subcloning technique. Finally, by binding two beta-galactosidase protein subunits and breaking down x-gal, soluble strains of TG2 protein were selected by means of blue color.In the other part of the project, non-mutant TG2 protein was recombinantly expressed and purified and its reactivity was investigated in promiscuous organic reaction syntheses, including the transamidation reaction, which was studied between two acyl donor substrates and eight acceptor substrates and reactions using Nessler test. The synthesis of dabsyl-putrescine molecule was afforded also performed to study the normal activity of the enzyme
  9. Keywords:
  10. Directed Evolution ; Glutaminase ; Trans Amidation ; Molecular Protein Engineering ; Recombinant Protein

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