Genetic Engineering of Suicide Genes to Improve Therapeutic Efficacies
| dc.contributor.author | Kohila, V | |
| dc.date.accessioned | 2015-09-21T07:15:40Z | |
| dc.date.accessioned | 2023-10-19T11:08:01Z | |
| dc.date.available | 2015-09-21T07:15:40Z | |
| dc.date.available | 2023-10-19T11:08:01Z | |
| dc.date.issued | 2013 | |
| dc.description | Supervisor: Siddhartha Sankar Ghosh | en_US |
| dc.description.abstract | Gene Directed Enzyme Prodrug Therapy, also known as Suicide gene therapy (SGT) has the potential to provide specific targeting of the tumor cells thereby conquer the deteriorate effects of chemotherapy treatment of cancer. As the inefficient binding of the prodrug(s) at the active site of the key enzymes which are involved in the SGT applications, the present thesis focused mainly on the development of novel suicide gene mutants to boost up their therapeutic efficacies. Bacterial expression vector which carries the gene codes for E. coli cytosine deaminase (CD) was constructed with the fused N-terminal GST tag. The single step purification of the recombinant enzyme was demonstrated and the functional activity assays with the substrates cytosine and 5-Fluorocytosine (5-FC) were examined. Further, the kinetic parameters were determined with double reciprocal plots. Emergence of novel CD mutants having enhanced binding affinity towards the prodrug than its actual substrate cytosine was elucidated with in silico site-directed mutagenesis. The study offered five mutants such as F186W, F82C, S126N and R91T which displayed efficient binding towards 5-FC as compared to cytosine. The study further revealed the explicit binding of the prodrug 5-FC with the mutant S126R. The in silico derived mutants were constructed in vitro; overexpressed, purified and further examined for their catalytic efficiencies towards the substrates cytosine and 5-FC. The investigation of functional characterization of these mutants revealed the fact that the redesigned proteins S126R, F186W and F82C exhibited enhanced catalytic efficiency towards the prodrug relative to the wild-type enzyme and could act as more potent candidates for SGT. Further in vitro studies showed drastic decrease in cell viability with increasing concentrations of prodrug 5-FC with the mutant S126R as compared to that of wild-type CD in human cervical cancer cell line, HeLa. In the next part, the construction of bacterial expression vector possesses the gene codes for the key enzyme E. coli Uracil phosphoribosyltransferase (UPRT) was demonstrated. Purification and preliminary functional assays of the recombinant enzyme with the substrates uracil and 5-Fluorouracil (5-FU) were performed. Like CD, the UPRT enzyme too has the similar constraint in binding with the drug 5-FU than the native substrate uracil. | en_US |
| dc.identifier.other | ROLL NO. 08610601 | |
| dc.identifier.uri | https://gyan.iitg.ac.in/handle/123456789/425 | |
| dc.language.iso | en | en_US |
| dc.relation.ispartofseries | TH-1197; | |
| dc.subject | BIOSCIENCES AND BIOENGINEERING | en_US |
| dc.title | Genetic Engineering of Suicide Genes to Improve Therapeutic Efficacies | en_US |
| dc.type | Thesis | en_US |