Browsing by Author "Yata, Vinod Kumar"

Now showing 1 - 1 of 1
  • No Thumbnail Available
    Item
    Suicide Enzymes : Purification, Characterization and Encapsulation for Therapeutic Implications
    (2011) Yata, Vinod Kumar
    Suicide gene therapy has been extensively investigated as an alternative approach to increase selectivity in cancer treatment. The strategy involves transfer of suicide gene to tumor cells followed by administration of prodrug, in which the transgene encodes enzyme that activates the prodrug to create toxic products and there by kill cancer cells. In addition, direct delivery of encapsulated suicide enzyme to cancer cells is another recent approach to activate prodrug. Two main themes of my thesis focus on purification, characterization of the suicide enzymes and development of nanocarriers encapsulating suicide enzyme. Non-mammalian cytosine deaminase (CD) catalyzes conversion of nontoxic prodrug, 5-fluorocytosine (5-FC) into the anticancer drug, 5-fluorouracil (5-FU). Therefore, 5- FC/CD mediated suicide gene therapy has strong potential in clinical application. In my thesis work, I have purified the suicide enzyme, cytosine deaminase to homogeneity from E. coli K-12 MTCC 1302 by fast performance liquid chromatography (FPLC) using DEAE-cellulose anion exchange column. I have characterized the enzyme and interestingly found that the enzyme was thermostabile at high temperatures. The enzyme was also active at a wide range of pH (7 to 10). Furthermore, E. coli uracil phosphoribosyltransferase (UPRT) enzyme is known to convert 5FU into active metabolite, 5-fluorouracil monophospahte (5FUMP) which kill the cells by inhibiting DNA and RNA synthesis. Therefore, the combine use of CD and UPRT generates very efficient gene therapy system. I purified UPRT from the same E. coli K-12 MTCC 1302 strain and studied inhibition kinetics to determine enzyme...