Pannarase Salt Active Nuclease is a non-specific nucleic acid endonuclease, which is expressed and purified from E. coli strain BL21. The protease is a single-chain polypeptide consisting of 149 amino acids with a molecular weight of 16.8 kDa, and its conformation can be stabilized by Ca2+ and Mg2+. This enzyme can degrade any form of DNA and RNA (linear, cyclic, superhelical) into 3'-mononucleotides and dinucleotides, and can remain efficient under a wide range of operating conditions. It can be used for downstream purification of viruses under high salt concentration and relatively alkaline conditions.
Properties:
Item | Description |
Specification | 100,000/500,000/5,000,000 U/tube |
Molecular weight | 16kDa |
Purity | ≥99% (SEC-HPLC) |
Specific activity | ≥2x106 U/mg |
Optimal pH | 8.5-10 |
Optimal temperature | 37°C |
Cofactor | 0.55-5 mM Mg2+；0.5-10 mM Ca2+ |
Storage temperature | -20°C, do not freeze and thaw repeatedly (It does not freeze at -20°C due to the presence of glycerin) |
Definition of active unit: when cleaving herring sperm DNA as the substrate (herring sperm DNA, Sigma, catalog number D7290) at 37°C, pH 8.0, one active unit of Pannarase Nuclease will produce a decrease of 1.0 in A260 (absorbance value at 260 nm) per 30 minutes (equivalent to a complete cleavage of 37 μg DNA).
Features:
Outstanding unit specific enzyme activity
Non-His label purification
Mass spectrum uniformity certification
Animal-origin free (AOF)
Suitable for high salt and alkaline conditions
Characterization by SDS-PAGE:
Non-reduced and reduced SDS-PAGE showed protein molecular weight of about 16 kDa with no protein impurity produced.
SEC-HPLC purity determination:
Size exclusion chromatography showed high purity with no aggregates produced
Application case study:
1. Effect of Na+ ions on the enzyme activity of Pannarase
Pannarase showed high activity at medium Na+ concentration, maintained 36.2% activity at 300 mM, but enzyme activity was almost completely lost at concentrations above 600 mM.
2. Effect of Mg2+ ions on the enzyme activity of Pannarase
1 mM Mg2+ boosts the activity of Pannarase endonuclease by about 40%, but Mg2+ is not indispensable
3. Effect of Ca2+ ions on the enzyme activity of Pannarase
1 mM Ca2+ boosts Pannarase endonuclease activity by 28%, but Ca2+ is not indispensable
4. Comparison of Pannarase enzyme activity under different pH conditions
The optimal pH for Pannarase enzyme activity was pH 8.0 - 11.0
Transportation and Storage:
Transport on dry ice. When stored at -15℃ ~ -25℃, the validity period is 2 year.
Recommended usage conditions:
Conditions | Optimal conditions* | Effective condition # |
Mg2+ | 0.55-5 mM | 0-10 mM |
Ca2+ | 0.5-10 mM | 0-10 mM |
pH | 8.5-10 | 7.0-11.0 |
Na+ | 0-150 mM | 0- 500 mM |
*“Optimal condition” means the condition under which Pannarase nuclease maintains >90% activity
**“Effective condition” means the condition under which Pannarase nuclease maintains >15% activity
Note: Please refer to the section "Pannarase product performance" for detailed parameters
Usage:
1.For fragmentized liquid of E. coli
For the purpose of viscosity reduction, the amount of nuclease to be added must be decided according to the concentration of bacteria in the fragmentized liquid. For the concentration of bacteria at 50%, the recommended amount of nuclease to be added is 1:1000-5:1000, i.e. 500,000-2.5 million U/L. For the concentration of bacteria at 5%, addition can be made in the ratio of 1:10,000 to 5:10,000.
2.For the cell lysate
Addition of 500 units of nuclease can be made for every 106-107 cells.
3.For purified adenovirus or viral vaccine
Due to the relatively small amount of DNA, it can be added at a final concentration of 0,01% to 0,05%, that is, 5 units per mL.
Attention:
1.This enzyme works best at pH 8.0-11.0, 37°C, for 30 min.
2.Due to the stable activity, it is appropriate to place the enzyme at room temperature for a short period of time, but it should be placed in the refrigerator at -20°C for long-term storage. The enzyme should be used as soon as possible after dilution with glycerol-free buffer, and it is not recommended to use it after frozen storage.
3.For the sole purpose of viscosity reduction, nuclease can be added directly and stirred slowly for 30 min at room temperature.
Order Imformations：
Cat.NO. Name Grade Size CY006F0010 Pannarase Salt Active Nuclease RUO 100 kU CYG006F0010 Pannarase Salt Active Nuclease GMP 100 kU CYG006F0050 Pannarase Salt Active Nuclease GMP 500 kU CYG006F0500 Pannarase Salt Active Nuclease GMP 5000 kU
Q:How to inactivate or remove Pannarase Salt Active Nuclease after use.
A:Due to the high isoelectric point of SAN (9.6), enzymes can be easily removed using ion exchange chromatography. Heat inactivation is not very effective. At 70 ° C, the half-life of SAN is about 2 hours, but SAN is susceptible to reducing agents such as DTT or TCEP, which can enhance enzyme thermal inactivation. Adding chelating agents can also inhibit SAN activity.
Q:Can Pannarase Salt Active Nuclease be used for the purification of viral vectors, such as AAV based vectors?
A:Yes, this endonuclease is suitable for purification of viral vectors. The purity of viral vectors is crucial before transduction in cells or animals. Salt is usually an important component of highly purified carriers by minimizing aggregation and increasing target production. High quality SAN is highly compatible with high salt conditions.
Q:What are the advantages and disadvantages of SAN compared to non-salt active nucleases?
A:Many protein buffers contain high concentrations of salt to release DNA from proteins. Compared with other nucleases, SAN exhibits the best activity at high salt concentrations. Because the optimal activity depends on salt, the activity of SAN may not be high when the salt content in the sample to be tested is very low.
Q:Is SAN active in the solution containing 0.1% of Triton® X-100
A:Yes. However, the activity will decrease to 70% of normal activity. At 0.5% of Triton ® , there will be almost no activity.
Q:Is SAN active in buffer containing 10% glycerol?
A:There is activity which has decreased, about 80% of the activity compared to samples without glycerol. In a 50% glycerol solution, there is almost no activity.
Q:Can SAN be used to replace all application scenarios of Benzonase?
A:In the application of non-specific nucleases that require action in high salt buffer, SAN can replace Benzonase.
Q:How to intuitively determine whether a SAN is functioning?
A:As SAN degrades DNA and RNA, the cell lysate becomes less viscous visually. Any nucleic acid testing method can be used to test the digestion effect of nucleic acids.
Q:What is the minimum reaction temperature for SAN?
A:The optimal reaction temperature is 37 ℃. SAN is also very active at low temperatures, but the reaction will be slower. The activity at 4 ℃ is approximately 5-10% of that at 37 ℃. This can be compensated by increasing the reaction time or the amount of enzymes used in the reaction.
