|Proteinase Kfrom yeast cells with cloned gene encoding Engyodontium album (Tritirachium album) endolytic protease|
|Catalog Number: GPK003001|
|CAS No.:39450-01-6||E.C.: 22.214.171.124|
|Synonyms: Peptidase K, Endoproteinase K, Endopeptidase K|
|Molecular mass||29.3 kDa|
|often used in pH range 7.5-9.0|
|Storage Temperature||-20 °C recommended|
|Form||The product is available as lyophilized powder and solution|
|Temperature profile||maximum activity at 70 °C|
|37-70°C temperature range recommended|
|Specific activity||≥ 34 units/mg of protein|
|Unit Definition: One unit is defined as the enzyme activity that produce1 µmol of tyrosine per minute from casein at 37°C at pH 7.5. Refer to the certificate of analysis for specific values for the present lot.|
|Extinction coefficient||E1% = 14.2|
|280 nm, 10 mM NaCl and 5 mM CaCl2, pH 8.0|
|Purity and Quality|
|DNA/RNA Free:DNA <1ppm, Quant-iT™ PicoGreen™ dsDNA
RNA <1ppm, Quant-iT™ RiboGreen RNA
RNase <0.001U/mg, Invitrogen RNaseAlert Lab Test Kit
Protein Content >95%
Cfu< 125/g, Total viable count testing
|General Description: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 is commonly used in molecular biology to remove protein contamination from preparations of highly native undamaged nucleic acid because it rapidly and effectively inactives nuclease that might degrade the DNA or RNA even in the presence of denaturing reagents.
Proteinase K is active in 1% Tritonä 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.
Advantages of Recombinant Proteinase K
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.
Gproan recombinant enzyme products are produced entirely from materials of synthetic origin and therefore are free from any animal derived materials including bovine products. In addition, there are no animal derived components used in the manufacturing or handling processes of Gproan recombinant enzymes. As such, Gproan recombinant enzymes can be declared free of Bovine Spongiform Encephalopathy (BSE) and Transmissible Spongiform Encephalopathy (TSE).
|Activators||1-5 mM Ca2+|
|To stimulate proteinase K activity, 1-5 mM Ca2+ can be added. Optimization using activators can increase proteinase activity significantly.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 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.|
|This product is soluble in water (1 mg/mL), yielding a clear colorless solution.Enzyme solutions prepared as recommended maintain stable at room temperature for 12 months.Dilution buffer recommended: 1mM calcium chloridem, 20mM Tris-HCl, pH 7.4;Storage buffer: 1mM calcium chloride, 20mM Tris-HCl, 50% Glycerol, pH 7.4.|
|Stability||storage at -20 °C|
|Gproan recommends long term storage at -20 °C for lyophilized powder to maintain stable for at least 3 years.|
|Precautions and Disclaimer|
|This product is for R&D use only, not for drug, household, or other uses.|