The HBV RNAse H has been cloned into the PET43a vector, which contains the NusA protein which works as a solubilizing fusion protein. The fusion NUS-RNAse H protein was cleaved by enterokinase; the cleaved RNAse H is about 17 Kda which remains soluble and active. A fluorescence assay utilizing a quenching mechanism was used to characterize the activity of NUS-RNAse H and cleaved RNAse H proteins. The beacon is a RNA:DNA hybrid oligonucleotide labeled with a 5'DABCYL and a 3'fluorescein, when RNAse H digests the RNA, DABCYL is released resulting in high fluorescence. The digestion of the RNA was also confirmed by gel analysis. The protein was identified by N-terminal amino acid sequence analysis of the fusion protein, SDS-PAGE, western blot utilizing HBV positive sera for primary antibodies, and enzyme immunoassay by peroxidase labeling of HBV RNAse H. Structural analysis of the protein was done by circular dichroism, tryptophan fluorescence, the generation of a model from HIV RNAse H and initial crystals which unfortunately did not diffract. The ability to produce good amounts soluble RNAse H, the development of a sensitive assay to test for activity and the solution of the crystal structure will help develop new anti-viral inhibitors.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd_retro-1015 |
| Date | 01 January 2007 |
| Creators | Saavedra, Mario Alejandro |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Retrospective ETD Collection |
| Rights | © The Author |
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