Proteinase K

Background

Proteinase K is commonly used for protein digestion, DNA extraction, RNA purification, direct PCR, and genotyping. Since it very effectively inactivates DNases and RNases, Proteinase K is added during nucleic acid preparations to isolate highly native, undamaged DNAs or RNAs; it is used in direct PCR kits for genotyping by destruction of proteins in cell lysates (tissue or cell culture cells) and for release of genomic DNA.

Proteinase K is a broad-spectrum serine protease originally isolated from fungus Engyodontium album. The protease was named “Proteinase K” for its ability to digest Keratin. Crystal and molecular structure studies suggest that the enzyme belongs to the subtilisin family characterized with a catalytic triad (Asp39-His69-Ser224) in active site. Proteinase K has no pronounced cleavage specificity and preferential cleavage site is the peptide bond adjacent hydrophobic amino acids.

Proteinase K exhibits higher proteolytic activity in the presence of reducing agents such as 5 mM DTT. Proteinase K is inhibited by serine protease inhibitors such as PMSF, DFP, and AEBSF.

Proteinase K is active in 1% Triton X-100 and fully active in 0.5% (w/v) SDS which denatures protein substrates to increase digestion rates. The enzyme works best at 50-200 ug/mL at pH 7.5-8.0, 37 °C and is usually denatured by subsequent phenol extractions. Incubation times vary from 30 minutes to 18 hours and proteinase K can auto-digest during long incubation.

The recombinant enzyme is a mutant to the native protease, which gains higher specific activity and yield as well as wider pH and temperature range . The large scale recombinant preparation has advantage in lot-to-lot consistency, superior purity and cost-efficiency. DNA-free nature of recombinant Proteinase K made it well-suited in isolating PCR and RT-PCR templates.

MB111: Proteinase K

The recombinant proteinase K enzyme is from yeast cells with cloned gene encoding Engyodontium album (Tritirachium album) endolytic protease.
CAS No.: 39450-01-6.
E.C.: 3.4.21.64.
Molecular Weight: 29.3 kDa.
pI: 8.9.
pH range: 4.5-12.0; often used in pH range 7.5-9.0.
Unit Definition: One unit of the proteinase K enzyme is defined as the enzyme activity that produce 1 µmol of tyrosine per minute from casein at 37°C at pH 7.5.
Specific activity: ≥ 34 units/mg of protein.
Temperature profile:  37-70°C temperature range recommended; maximum activity at 70°C.
Extinction coefficient:  E^1% = 14.2; 280 nm, 10 mM NaCl and 5 mM CaCl2, pH 8.0.
Formulation: The proteinase K enzyme is available as lyophilized powder and solution.
Reconstitution: The proteinase K enzyme is soluble in water (1 mg/mL), yielding a clear colorless solution.
Concentration: 500 unit/ul.
Purity: Each lot of the proteinase K enzyme was tested to ensure the absence of Nucleases and DNA. DNase is not detected in quality control procedure of incubation 40 µg Proteinase K with 1 µg λ DNA for 6 hours at 37°C. RNase is not detected in quality control procedure of incubation 40 µg Proteinase K with 2 µg RNA for 2 hours at 37°C. The preparation is considered as RNase and DNase free.

Solution preparation: The molecular biology grade lyophilized powder is NOT sterile.  The proteinase K stock solution can be prepared as a 20-40 mg/ml in 20 mM Tris-HCl buffer, sterilized using a 0.22 µm filter and supplied in 50% sterilized glycerol to store in aliquots at wide temperature range from 24°C to -80°C. PES or PVDF membranes with low protein binding are recommended in sterile filtration. We offer irradiation sterilization option to bulk quantity order. The Gamma irradiation procedure may cause slight enzyme activity loss. Please inquiry.

Activators: 1-5 mM Ca²⁺. To stimulate proteinase K activity, 1-5 mM Ca²⁺ can be added. Optimization using activators can significantly increase proteinase activity. Enzyme activity will be reduced by 25% when calcium is removed by addition of EDTA. Enzyme activity will be reduced by 80% if the EDTA-Ca²⁺ complex is removed from the enzyme solution by gel filtration, while it can be partially restored by addition of excess Ca²⁺.

Inhibitors: DIFP or PMSF. The proteinase K enzyme is inactivated by DIFP or PMSF (PMSF used at final concentration 5 mM.). However, it is not inhibited by EDTA , iodoacetic acid, trypsin-specific inhibitor TLCK, chymotrypsin-specific inhibitor TPCK, and p-chloromercuribenzoate.

Shipping: The proteinase K enzyme is shipped with ice packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
>2 years from date of receipt, -20 to -70°C for lyophilized powder.
Enzyme solutions prepared as recommended maintain stable at room temperature for 12 months. To prolong the shelf-life of Proteinase K, storage at 2–8°C is recommended.
Storage buffer: 20 mM Tris-HCl, 50% Glycerol, pH 7.4.
Dilution buffer recommended: 20 mM Tris-HCl, pH 7.4.

Questions and Answers:

Question: Can the proteinase K enzyme be used for direct PCR?
Answer: Yes. The enzyme can be used for isolation of genomic DNA from mouse tail and cultured cells, and removal of DNases and RNases when isolating DNA and RNA from tissues or cell lines.  A simple "Universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification.

Question: What is the recommended working concentration of the proteinase K enzyme?
Answer: The recommended working concentration of the proteinase K enzyme is 0.05 to 1 mg/mL. The enzyme activity is stimulated by 0.2 to 1% SDS or by 1 to 4 M urea. 

Question: Does the proteinase K enzyme work at room temperature?
Answer: The recommended temperature range for proteinase K digestion is 37-65°C; optimum activity at 50 to 55°C; maximum activity at 70°C and rapid denaturation of enzyme occurs at temperatures above 65°C.

Question: How to prepare proteinase K stock solution?
Answer: The most common proteinase K stock solution of 20 mg/ml is prepared by dissolving the lyophilized proteinase K powder in sterile water (stable for 1 year at −20°C) or in a solution of 50 mM Tris, pH 8.0, 1 mM CaCl2 (stable for months at 4°C).

Question: How to prepare proteinase K work solution?
Answer: Proteinase K is generally used at a working concentration of up to 50 μg/ml in various buffer formulations, including those that contain up to 0.5% SDS.

Question: How to inactivate proteinase K?
Answer: The most recommended proteinase K inactivation method is to heat the proteinase K enzyme to 95°C for 10 minutes.  However, the enzyme cannot be completely inactivated by heating. The subsequent washing is also recommended to remove the leftover enzyme.