| Abstract |
Type II restriction enzymes are the modern 'scissors' of molecular biology with various applications in biomedical research, biotechnology and diagnostics. Extensive studies in the second half of the last century have focused on their biological activity in bacterial cells, while the revolution of recombinant DNA technology was the means to further research on the structure-function relationship of these enzymes. Among them, the restriction enzyme EcoRV is one of the most well-studied proteins, and PvuII, one of the smallest type II restriction endonucleases, has been the subject of numerous structural studies. Although lacking significant homology in their primary sequence, these two enzymes exhibit remarkable structural similarity. This was the driving force for the creation of an artificial fusion protein, designed to perform a new specialized endonucleolytic digestion reaction, within the framework of engineering innovative enzymes. Τhe single chain protein EcoRV-PvuII incorporates the genetically engineered protein domains of the restriction enzymes EcoRV and PvuII. The EcoRV-PvuII protein was designed in the Crystallography laboratory of Prof. Kokkinidis and its production and study was the subject of the present thesis. The initial aim was the expression of the protein in a soluble form, since restriction enzymes normally exhibit their biological activity in solution. Subsequently, the protein was isolated, in order to use it for digestion assays, with plasmid DNA as a substrate, and potential structural characterization. Using Affinity Chromatography, the protein was isolated effectively from a NiNTA agarose column in the presence of denaturing agents, since it forms inclusion bodies. However, due to non-specific activity observed in the digestion assays with DNA, a new experimental protocol was employed that allowed for the partial purification of the protein partial purification was performed in non-denaturing conditions. Surprisingly, electrophoretic analysis revealed visible improvement on the specific activity of EcoRV-PvuII, but it resembles greatly the specificity of the restriction endonuclease PvuII. This, rather unexpected, result calls for further investigation of the EcoRV-PvuII protein, and, if it can be isolated more efficiently under non-denaturing conditions, Crystallographic analysis experiments with X-rays. The specificity of EcoRV-PvuII can be established more thoroughly through digestion test experiments with oligonucleotide DNA substrates, which are in progress.
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