molecular biology Proteinase K (also protease K or endopeptidase K) is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Engyodontium album (formerly Tritirachium album). Proteinase K is able to digest native keratin (hair), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8. The molecular weight of Proteinase K is 28,900 daltons (28.9 kDa).
| During the extraction of DNA (or nucleic acids in
general), there is a lot of contaminating proteins present. These
contaminants must be removed. Proteinase K, which is a broad spectrum
serine protease, is used in many DNA extraction protocols to digest
these contaminating proteins.
In addition, there may be nucleases (enzymes that degrade nucleic
acids) present. The addition of proteinase K degrades these nucleases
and protects the nucleic acids from nuclease attack. In addition,
proteinase K is stable over a wide pH range and is well suited for use
in DNA extraction.
proteinase K applications?
Proteinase K is commonly used in molecular biology to
digest protein and remove contamination from preparations of nucleic
acid. Addition of Proteinase K to nucleic acid preparations rapidly
inactivates nucleases that might otherwise degrade the DNA or RNA during
purification. It is highly-suited to this application since the enzyme
is active in the presence of chemicals that denature proteins, such as SDS and urea, chelating agents such as EDTA,
sulfhydryl reagents, as well as trypsin or chymotrypsin inhibitors.
Proteinase K is used for the destruction of proteins in cell lysates
(tissue, cell culture cells) and for the release of nucleic acids, since
it very effectively inactivates DNases and RNases. Some examples for
applications: Proteinase K is very useful in the isolation of highly
native, undamaged DNAs or RNAs, since most microbial or mammalian DNases
and RNases are rapidly inactivated by the enzyme, particularly in the
presence of 0.5 - 1% SDS. Purification of genomic DNA from bacteria
(miniprep): bacteria from a saturated liquid culture are lysed and
proteins are removed by a digest with 100 μg/ml Proteinase K for 1 h at
37 °C. The enzyme's activity towards native proteins is stimulated by
denaturants such as SDS. In contrast, when measured using peptide
substrates, denaturants inhibit the enzyme. The reason for this result
is that the denaturing agents unfold the protein substrates and make
them more accessible to the protease.