In protein studies DNA often poses a problem as it creates viscosity in the sample and interferes with downstream protein analysis. To limit protein-DNA interactions, the ionic strength may be raised by the addition of salts. However, even at moderately high salt concentration most nucleases become inactive, making enzymatic removal of DNA problematic. We have recombinantly expressed a Salt Active Nuclease (SAN) isolated from a marine psychrophilic bacterium in the yeast Pichia pastoris. SAN is a highly active non-specific endonuclease that cleaves both DNA and RNA. The enzyme has an optimum activity at 0.5 M NaCl and is active in the pH range of 7-10. It has a temperature optimum around 37Â°C, and retains significant activity also at low temperatures. The enzyme is stable upon moderate heat treatment with a half-life of 5 hours at 60Â°C, and is stable for weeks at room-temperature. We have tested SAN for use in removal of DNA from Escherichia coli lysates, and show that about 1μg of enzyme efficiently removes DNA from a 0.1 ml sample in 30 minutes. The tolerance for high salt concentrations and low reaction temperature make the new Salt active nuclease advantageous for use in removal of DNA from cell extracts and protein samples.
Full conference title:
110th General Meeting American Society for Microbiology
- ASM 110th (2010)