Endonucleases Non-Specific

Endonucleases Non-Specific, HL-SAN

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HL-SAN efficiently removes nucleic acids from buffers typically used in protein purification. Due to its high salt tolerance, it is the obvious choice for host-cell DNA removal in settings where salt is added to reduce aggregation. Especially efficient for removing nucleic acids from proteins with high affinity for DNA and RNA. Proven performance during lysis and early stages of protein purification processes, as well as high-salt eluates. HL-SAN is a cold-adapted enzyme with excellent performance also at ambient temperatures and during over-night digestion at 4°C.

Recommended applications:

Complete removal of DNA

Viscosity reduction

Key advantages with HL-SAN:

Non specific endonuclease
Optimum activity at high salt concentration (0.5 M NaCl)
Active at low temperatures (20% at 6ºC)
Easily inactivated
Broad pH range
Temperature stable

Figures

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.

Fig 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.

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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%.

Properties

Source

Recombinantly produced in Pichia pastoris.

Working Ranges

Temp: 7 – 38°C and 4°C overnight, optimal: 30 – 38°C
Salt concentration (NaCI / KCI): 0 - 1000 mM, optimal: 400 - 700 mM
Mg₂₊: >1 mM is required for activity, optimal: 5 – 50 mM
pH: 6.8 - 9.5, optimal: 8.0 - 8.9

Note: The working range is defined as above 10% of activity and optimal range as above 80% activity.

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.

Tolerance to Typical Lysis Buffer Additives

Imidazole: 20% activity at 350 mM Imidazole
Glycerol: 20% activity at 35% glycerol
Triton X-100: No reduction in activity in Triton X-100 (tested up to 15%)
Tween20: No reduction in activity (tested up to 10%)
SDS: Not recommended
Urea: Not recommended
Reducing agent (e.g. DTT, TCEP): will result in inactivation

Quality Control

ArcticZymes is dedicated to the quality of our products. HL-SAN is manufactured at our ISO 13485 certified facility in Norway.

dsDNA Endonuclease Activity

25 U IsoPol® BST+ was incubated with a supercoiled plasmid (1 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any transformation of closed circular DNA to nicked DNA.

ssDNA Endonuclease Activity

25 U IsoPol® BST+ was incubated with M13 ssDNA (0.5 µg) for 4 hours at 37°C. Agarose gel electrophoresis did not reveal any visible signs of ssDNA degradation.

Recommended Protocols

Ordering Info

Quantity

Product Name

SKU

Pack Size

Concentration

Price

T7 RNA Polymerase

74100-201

10 000 U

50 U/µl

$ 145.00 USD

Endonucleases Non-Specific