Protein Purification

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. Nucleases activity is usually difficult to remove while HL-SAN is easily inactivated or separated from other proteins. This enables nuclease treatment without residual nuclease activity in downstream applications.

HL-SAN is easily inactivated by treatment with a reducing agent, and the high pI (9.6) enables easy separation of HL-SAN from a vast majority of protein targets. The optimal activity at high salinity and the resistance to non-ionic detergents enable HL-SAN treatment at conditions facilitating dissociation of DNA from DNA-protein complexes to make it more accessible for degradation. These features make HL-SAN the superior choice for DNA digestion in your protein purification workflow.

Learn more about HL-SAN →

Guidelines for DNA removal

The amount of HL-SAN needed for DNA removal from a cell extract or lysate depends on several factors; expression strain, target protein, lysis buffer composition, NaCl concentration, etc. The following is therefore considered as guidelines: Add 1000 U HL-SAN per ml sample with 0.3–0.75 M NaCl and incubate at 15–37°C for 30–60 minutes or at 4°C overnight. Mg is required for activity.

DNA may cause a problem during protein purification and in the final product. In the first steps of a purification scheme, only fragmentation of genomic DNA in the lysate/extract is usually necessary. However, even small amounts of DNA can result in a contaminated product and using HL-SAN in later steps in the protein purification workflow will facilitate removal of traces of nucleic acids (decontamination).

Recommended operating conditions

Condition Optimal Effective*
Salt (NaCl/KCl) 500 mM 50 mM-1 M
Temperature ~35°C 4-50°C
Mg2+/Mn2+ 5-20 mM 1-40 mM
pH 9.0 7.0-9.5

*Effective is defined as the condition which HL-SAN has ≥ 10 % of its activity as compared to optimal conditions.

Inactivation

Inactivation is achieved by adding reducing agents like TCEP or DTT, where TCEP* is the recommended reducing agent. The inactivation protocol can be adapted to several workflows by varying incubation time, temperature and concentration of the reducing agent. In general >99% inactivation is achieved after 5–10 minutes at 25–37°C.
To avoid reactivation, maintain a low concentration of reducing agent, 0.1–0.5 mM DTT or TCEP, or use prolonged incubation times with 10–20 mM TCEP upon inactivation.

*Low stability in phosphate buffers, especially at neutral pH.

Guidelines for inactivation

Temperature/Time DTT TCEP
4°C/18 hours 10 mM
25°C/60 minutes 10 mM 5 mM
30°C/30 minutes 10 mM 5 mM
40°C/30 minutes 5 mM 1 mM
50-70°C/30 minutes 1 mM 1 mM

Removal

The very high pI (9.6) of HL-SAN results in tight binding to cationic columns. Even at pH 9.0 with 0.2M salt, less than 0.02% leakage in flow-through/wash is observed. It is not recommended to use anionic IEX columns for removal of HL-SAN as the glycosylation of HL-SAN result in column binding.

Conditions for binding HL-SAN to SP-sepharose

pH Salt
pH 7 ≤ 0.3 M
pH 8 ≤ 0.3 M
pH 9 ≤ 0.2 M
HL-SAN binds tightly to SP-sepharose columns
Figure 1: HL-SAN binds tightly to SP-sepharose columns at pH 9.0 with 0.2M salt (less than 0.02% leakage).

DNA removal from various samples

Sample HL-SAN final concentration Recommended conditions
DNA removal* Decontamination**
Protein 100 U/ml 1000 U/ml 30 minutes at 25–37°C
Reagent 100 U/ml 1000 U/ml 30 minutes at 25–37°C
Cell extract 1000 U/ml N/A 60 minutes at 25–37°C or 4°C overnight
Cell lysate (soluble fraction) 500 U/ml N/A 60 minutes at 25–37°C or 4°C overnight
Viscosity reduction 25-50 U/ml 10-20 minutes at 25°C

* DNA amount is reduced to a level that cannot be detected by visualization using agarose gel electrophoresis.** DNA amount is reduced to a level generally not detectable by a 23S rDNA qPCR assay.

Properties

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.