AbstractIn addition to the bacteria and eukarya, archaea were suggested as the third domain of life by Woese & Fox, 1977. As with viruses infecting eukaryotes and bacteria, archaeal viruses are seen across the archaeal spectrum infecting halophiles, thermophiles and methanogens. Viruses infecting extremophilic archaea have evolved machineries that enable them function at extreme temperature, salinity, and pH that can make them biotechnologically useful. Genomic sequencing had revealed that some archaeal viruses encode putative family B DNA polymerase. In this study, type B DNA polymerase genes encoded by Methanosarcina Spherical Virus 1 (MetSV) and Sulfolobus ellipsoid virus 1 (SEV1) were cloned into various N-terminal 6xHis-tag and C-terminal 6xHis/FLAG-tag pET expression vectors using ligase independent cloning. Small-scale expression of these DNA polymerases resulted in insoluble proteins. N-terminally and C-terminally tagged constructs encoding family B DNA polymerases from halophilic archaeal viruses His1 and HF2 were also expressed in small scales for optimisation. His1 DNA polymerase protein expression at large scales using chromatography did not yield sufficient amount of protein to perform enzyme assays. However, expression and purification of N-terminally and C-terminally tagged HF2 DNA polymerase at large scales using the same techniques gave sufficient amount of soluble protein for enzyme assays. Polymerase and exonuclease titration assays were performed at different salt (NaCl/ KCl) concentrations and temperature. Denaturing PAGE analysis of assays products revealed C-terminal tagged HF2 DNA polymerase synthesis and exonuclease activities at lower salt concentrations and incubated at higher temperatures (45ºC). Multiple sequence alignments confirmed the presence of conserved motifs for 3'-5' exonuclease (DxE) and DNA polymerase
(Dx2SLYP / YxDTDS ) in HF2 DNA polymerase suggesting it is likely to be an active enzyme. However, it is essential to validate both HF2 DNA polymerase identity and its activity in future studies.
|Date of Award||2023|
|Supervisor||Michael Ginger (Co-Supervisor) & Chris Cooper (Co-Supervisor)|