Loading...

Cloning and Production of the Flavin Reductase Enzyme in Order to Perform Organic Reaction and Synthesis of Cation Exchange Column for Protein Purification

Hayati, Fatemeh | 2020

527 Viewed
  1. Type of Document: M.Sc. Thesis
  2. Language: Farsi
  3. Document No: 53968 (03)
  4. University: Sharif University of Technology
  5. Department: Chemistry
  6. Advisor(s): Kalhor, Hamid Reza
  7. Abstract:
  8. The reductase enzymes perform an important function in the cell. These enzymes are usually coupled with other enzymes and participate in reactions such as halogenation, reduction of alkene, and carboxylic acids. These reactions are carried out by reproducing cofactors such as FADH2 and NAD+; however, there is no evidence that this enzyme alone can perform an organic reaction. In this work, we aim to examine whether recombinant flavin reductase can carry out organic reactions. In order to amplify the flavin reductase enzyme gene from the E. coli genome by PCR (Polymerase Chain Reaction) method, two synthetic primers were designed; The forward primer of the gene contained a cleavage site for the NdeI enzyme and the reverse primer contained the cleavage site for the XhoI enzyme. First, the bacterial cell was grown and its genome was extracted. Using PCR method and two designed primers, the flavin reductase gene was amplified, and the product was cloned in to the vector containing the antibiotic kanamycin. The ligated reaction between PCR product and the vector was transferred into the bacterial cells and successful clones were selected using kanamycin-containing media; after confirmation the correct clones, recombinant protein flavin reductase was expressed in using IPTG inducer. Using SDS PAGE electrophoresis, it appeared that the expressed protein was mainly in the particulate fraction. Subsequently the expressed protein was solubilized and was refolded. The activity of recombinant flavin reductase was evaluated by using riboflavin and NADH, monitoring hydride transfer at 340nm. Ultimately using recombinant flavin reductase, a number of reducing organic reactions were examined. The reduction of carbonyl compounds and azo compounds were used in different conditions and the possibility of getting a new product was checked using TLC. The preliminary results indicated that under conditions examined the flavin reductase enzyme was not able to carry out the reduction reactions of carbonyl compounds; but in the case of Azobenzene, TLC results showed that this enzyme could reduce azobenzene to Biphenyl instead of aniline, and C-C coupling (Two Phenyl rings) was done with the release of nitrogen molecule.Protein purification columns are widely used in the laboratory, and due to the high cost of purification columns, their synthesis in the laboratory will be very valuable in terms of cost savings. In this work, we want to synthesize a cationic column that is specifically used to purify positively charged proteins using biodegradable polymers such as agarose, chitosan, carboxymethylcellulose, sodium alginate, a polymer substrate was made that should be used to separate positively charged proteins due to its negatively charged functional groups at its surface. The product was examined using FTIR at each step in the synthesis. Finally, lysozyme protein, which contains a large number of positively charged amino acids, was used to test the binding of a protein to a cationic column. The results demonstrated that the synthesized column did display binding for lysozyme, but it did not release the protein bound completely
  9. Keywords:
  10. Protein Purification ; Flavin Reductase Enzyme ; Cationic Column ; New Organic Reaction ; Cloning ; Polymerase Chain Reaction (PCR)

 Digital Object List

 Bookmark

No TOC