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.
Active at high salt conditions
|Source||Recombinantly produced in Pichia pastoris.|
|Activity||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.|
|Specific activity||≥ 175 000 Units/mg|
|Unit definition||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.|
Figure 1: Optimum activity in solutions with high salinity. HL-SAN has optimum activity at ∼0.5 M NaCl, but operates at a broad range of [NaCl] and [KCl]. The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2 with varying [NaCl] or [KCl]. The maximum activity was set to 100%.
Figure 2: Temperature and activity. HL-SAN has optimum activity at ~35°C, but works over a broad temperature range (20% activity at 10°C and 50°C). The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5 containing 5 mM MgCl2 and 0.5 M NaCl.
Figure 3: The effect of MgCl2 and MnCl2 concentration on the HL-SAN activity.
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 0.5 M NaCl and with varying concentrations of MgCl2 or MnCl2. The activity of the sample containing 5 mM MgCl2 was set to 100%.
Figure 4: HL-SAN activity vs pH/[NaCl]. The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer with different pHs and different concentrations of NaCl. All buffers contained 5 mM MgCl2. The nature of the buffer was pH-dependent, but generally the NaCl-optimum was the same in all buffers/pHs. The exception was etanolaminbuffer at pH 9 and pH 9.5 in which the NaCl-optimum was shifted to the left (not shown).
Figure 5: Buffer composition affects substrate preference. Without NaCl, the specificity towards ssDNA and dsDNA is similar. At 0.5 M NaCl, the activity towards dsDNA increases, while the activity towards ssDNA is unaffected
Figure 6: HL-SAN digests ssDNA to ~5-13 nt, and dsDNA to ~5-7 nt. The size of the end products from ssDNA varies from ~5-13 nt, while dsDNA is digested to around ~5-7 nt. The size of the end products seems to depend on the DNA sequence. Substrates 1 and 2 were ssDNA with different sequences and substrates 3 and 4 were dsDNA with similar sequences but with a FAM-label at different ends. Substrate 5 was dsDNA with the same sequence as substrate 3 and 4 but with a FAM-label at both ends
Figure 7: HL-SAN activity decreases with increasing concentrations of glycerol.
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with increasing concentrations of glycerol. The activity of the control not containing glycerol was set to 100%.
Figure 8: The activity of HL-SAN at different concentrations of imidazole.
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with varying concentrations of imidazole. The activity of the control not containing imidazole was set to 100%.
Tolerance to typical lysis buffer additives
|Imidazole||20% activity at 350 mM Imidazole|
|Glycerol||20% activity at 35% glycerol|
|Triton X-100||No reduced activity in Triton X-100 (tested up to 15%)|