Poly(ADP-ribosylation) is a post-translational covalent modification of proteins and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catalyzed by a family of enzymes termed poly(ADP-ribose) polymerases (PARPs). TcPARP of Trypanosoma cruzi, appears to play a role in DNA repair mechanism during Chagas’ disease and believed to be involved in controlling different phases of cell growth. In this study, cloning and characterization of mutants of TcPARP is reported. In first phase, five mutants of TcPARP, already cloned in pET-22b+, were expressed BL21 Rosetta2(DE3). Since expression in this vector was poor, all mutants were cloned in pNH-TrxT vector and expressed in BL21 Rosetta2(DE3). Protein purification of mutant TCP-c006 (124 residues deleted from N-terminus) was performed using His6 tagged FF crude IMAC column followed by gel filtration. Fluorescence-based activity assay of mutant TCP-c006 shows only 35% conversion of NAD+ to nictonamide compared to wild type. Moreover 50 times more protein is required for this conversion when compared with wild type. During DNA binding assay (EMSA), mutant TCP-c006 did not bind with DNA, clearly indicating that N-terminus is necessary for DNA binding and activity of TcPARP.
In future, solubility of both wild type and mutants of TcPARP in different expression host system using various fusions such as MBP, T4 lysozyme etc could be tested. Structure of both wild type and mutants of TcPARP could be determined by using Static light scattering (SLS).
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:nbnfioulu-201309131688 |
| Date | 16 September 2013 |
| Creators | Nosheen, S. (Saman) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © Saman Nosheen, 2013 |
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