Recombinant Proteinase K, 100 mg lyophilized

Proteinase K (EC 3.4.21.64) is an extracellular serine endopeptidase initially discovered from fungus Tritirachium album Limber. Proteinase K belongs to the peptidase family S8, and its predominant cleavage site is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked L-amino groups ^{[1] [2]}. Because of its broad sequence specificity and stability in variant pH and temperature conditions, proteinase k is commonly used in biology sample processing. Proteinase K is stable over a wide pH range from 4-12, when pH is 8.0 ^[3], the activity reaches the peak, the activity also increases with elevated temperature from 37^oC to 60^oC. Some denature reagents extensively used in nucleic acid purification don%› inhibit proteinase K%š activity, such as 3M Guanidinium Chloride, 3M Guanidinium Thiocyanate, 1% SDS and 4M urea etc, which make proteinase K a favorite tool by molecular biologist in protecting RNA or DNA from degradation by nucleases, for example, proteinase K can be added to extraction buffer during nucleic acid preparation to cleave nucleases such as DNases and RNases, and preserve the nucleic acids from degradation.

For the time being COVID-19 pandemic is threatening human life, and has been causing huge damage globally, COVID-19 RNA identification by PCR is the golden standard for diagnosis, virus sample preparation is a key step in the diagnosis procedure, proteinase K is one of the important reagent to make sure that the target RNA is intact during the sample preservation and processing ^[4]. AmeriDx® Proteinase K has high purity (>98%) and great activity (>=30U/mg), and meet your requirement for virus detection.

Table 1. AmeriDx® Proteinase K

Proteinase K Assay Enzyme Activity is >= 30U/mg

Denatured Casein was mixed with protein K solution, incubated at 55^oC for 5 min, stop the reaction by TCA, after filtrating out the precipitate, determine the A275 absorbance value in the soluble fraction, and calculate the enzyme activity ^[1].

The standard curve used for this enzyme activity calculation shown in Figure 1, y-axis is the absorbance at 275nm (A275), x-axis is tyrosine concentration for each assay point.

Unit definition: One unit is defined as the amount of enzyme that catalyzes the formation of 1 µmol of tyrosine per minute at pH 7.5 and 37°C.

Fig.1 Standard Curve of L-tyrosine at 275 nm

Protein Purity – 98%

SDS-PAGE analysis of the AmeriDx® Proteinase K sample shows that protein is over 98% purity and the molecular weight is 28.9 kDa (Fig.2).

Fig.2 SDS-PAGE Image of AmeriDx® Proteinase K

AmeriDx® Proteinase K Is Stable Over One Year.

AmeriDx® Proteinase K lyophilized powder had been stored at 4oC for 12 months, its enzymatic activities were assayed every month, the activity still retains more than 95% (Fig 3).

Fig.3 The Stability Test of AmeriDx® Proteinase K at 4℃

References

1. Morihara K, Tsuzuki H. Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides. Agricultural and Biological Chemistry. 1975;39(7):1489-92. doi: 10.1271/bbb1961.39.1489.

2. Kraus E, Kiltz HH, Femfert UF. The specificity of proteinase K against oxidized insulin B chain. Hoppe-Seyler's Zeitschrift fur physiologische Chemie. 1976;357(2):233-7. Epub 1976/02/01. PubMed PMID: 943367.

3. Ebeling W, Hennrich N, Klockow M, Metz H, Orth HD, Lang H. Proteinase K from Tritirachium album Limber. Eur J Biochem. 1974;47(1):91-7. Epub 1974/08/15. doi: 10.1111/j.1432-1033.1974.tb03671.x. PubMed PMID: 4373242.

4. de Paula VS, Villar LM, Coimbra Gaspar AM. Comparison of four extraction methods to detect hepatitis A virus RNA in serum and stool samples. The Brazilian journal of infectious diseases : an official publication of the Brazilian Society of Infectious Diseases. 2003;7(2):135-41. Epub 2003/09/10. doi: 10.1590/s1413-86702003000200007. PubMed PMID: 12959685.