Removal of nucleic acids may improve workflows during protein purification, both for laboratory sample preparation and industrial bioprocessing. HL-SAN is a nonspecific endonuclease with optimum activity at high salt concentrations. HL-SAN is active in a variety of buffers and can be easily inactivated by treatment with a reducing agent. These features make HL-SAN particularly useful in the purification of proteins and removal of DNA and RNA from molecular biology reagents.
Nucleic acids, and especially genomic DNA, often pose a problem in purification of DNA-binding proteins as they interfere with purification, downstream analysis or applications.
As most nucleases are inhibited by high concentrations of salt, removal of DNA is difficult, since NaCl is used to dissociate DNA from proteins.
The high salt-tolerance and easy removal makes HL-SAN beneficial to use in protein purification schemes, especially in combination with IMAC (Immobilized metal affinity chromatography) media used for purification of poly-His-tagged proteins both in conventional chromatography or high-throughput settings.
|Recombinantly produced in Pichia pastoris.
|HL-SAN is highly active in the temperature range 10–50°C. Optimal NaCl-concentration for activity is 0.5 M, working range is 0.25–1 M. Mg2+ (>1 mM) is required for activity. Working pH range is 7.5–9.5, optimal pH is 9.0.
|≥ 175 000 Units/mg
|One Unit is defined as an increase in absorbance at 260 nm of 1 A in 30 minutes at 37°C, using 50 μg/ml calf thymus DNA (D-1501, Sigma) in a buffer consisting of 25 mM Tris-HCl, pH 8.5 (25°C), 5 mM MgCl2, 500 mM NaCl.
|20% activity at 350 mM Imidazole
|20% activity at 35% glycerol
|No reduced activity in Triton X-100 (tested up to 15%)