Proteinase K

Background

What is proteinase K used for? Proteinase K is commonly used for protein digestion, DNA extraction, RNA purification, direct PCR, and genotyping. Since it very effectively inactivates DNases and RNases, the recombinant proteinase K enzyme 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. The proteinase K enzyme has no pronounced cleavage specificity and preferential cleavage site is the peptide bond adjacent hydrophobic amino acids.

The proteinase K enzyme 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 proteinase K 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 Powder

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; high activity in the pH range of 7.5-11.5.
Unit Definition: One unit of the proteinase K enzyme is defined as the enzyme activity that produce 1 umol 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.
Purity: >99.5% by reducing SDS-PAGE. Each lot of the proteinase K (powder and solution) 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 ug λ DNA for 6 hours at 37°C. RNase is not detected in quality control procedure of incubation 40 µg Proteinase K with 2 ug RNA for 2 hours at 37°C. The preparation is considered as RNase and DNase free.
Formulation: lyophilized powder.
Reconstitution: The proteinase K enzyme is soluble in water (1 mg/mL), yielding a clear colorless solution.
Solution preparation: The molecular biology grade recombinant proteinase K, lyophilized powder is NOT sterile. It can be used to make molecular biology grade proteinase K solution, PCR grade proteinase K solution, NGS grade proteinase K solution, CHI grade proteinase K solution, and so on. 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.
Storage buffer: 20 mM Tris-HCl, 1 mM CaCl2, 50% Glycerol, pH 7.4.
Dilution buffer recommended: 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2; or 20 mM Tris-HCl (pH 7.4), 1 mM CaCl2, and 2% Glycerol.
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 recombinant proteinase K powder 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 if stored at -20 to -70°C.
>1 years from date of receipt if stored at room temperature.
>1 years from date of receipt if stored at 2–8°C.

PCR enzymes:
* Taq DNA polymerase.
* High yield Taq DNA polymerase.
* Hot-start high-fidelity DNA polymerase.
* Hot start Taq DNA polymerase with chemical or antibody inhibitor, used for SYBR green or Taqman probe-based real time PCR (qPCR or quantitative PCR).

2x PCR master mixes:
* 2x PCR PreMix with dye (green, red, blue).
* 2x rapid PCR Master Mix with a green dye.
* 2x long PCR Master Mix with a blue dye.
* 2x high yield PCR master mix with a blue dye.
* 2x hot-start high-fidelity PCR master mix with a blue dye.
* Blood direct PCR kit
* Cell and tissue direct PCR kit
* RT-PCR master mixes
* 2x qPCR master mixes
* RT-qPCR master mixes

PCR components and related products:
* dNTP mix, 10 mM each.
* dNTP mix, 25 mM each.
* Proteinase K powder.
* Proteinase K solution.
* Reverse Transcriptases.
* First strand cDNA synthesis kits.
* Murine RNase inhibitor.
* PCR enhancer.

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: Can you provide any proteinase K buffer recipe?
Answer: Depending on the specific applications of the enzyme, the proteinase K buffer recipe may be different. Our recombinant proteinase K powder can be dissolved in water or storage buffer: 20 mM Tris-HCl, 1 mM CaCl2, 50% Glycerol, pH 7.4.

Question: Does the recombinant 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 make Proteinase K solution from powder?
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 20 mM Tris-HCl, 1 mM CaCl2, 50% Glycerol, pH 7.4 (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 ug/ml in various buffer formulations, including those that contain up to 0.5% SDS.

Question: How to inactivate the recombinant proteinase K?
Answer: The most recommended proteinase K inactivation method is to heat the recombinant 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.