What is proteinase K? What is its role in PCR technology?
Introduction to Proteinase KProteinase K is a powerful proteolytic enzyme isolated from Candida albicans with high specific activity and is a key reagent for DNA extraction.
Proteinase K is a serine protease with broad cleavage activity. It cleaves the carboxy-terminal peptide bonds of aliphatic and aromatic amino acids. The enzyme is purified to remove RNase and DNase activities. The general working concentration of proteinase K is 50-100 μg/ml. It is active in a wide pH range (pH 4-12.5) and is used for the isolation of plasmid or genomic DNA and RNA. In DNA extraction, the main function is to enzymatically hydrolyze the histone bound to nucleic acid, so that DNA is free in solution, and then different methods are used for extraction to remove impurities and collect DNA.
The origin of proteinase K
In molecular biology, proteinase K (also known as proteinase K or endopeptidase K) is a broad-spectrum serine protease. The enzyme was discovered in 1974 in an extract of the fungus Engyodontium album (previously Tritirachium album). Proteinase K digests natural keratin (hair), hence the name "Protease K". The primary cleavage site is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups.
Why is the Proteinase K enzyme used for DNA extraction?
Proteinase K is used during DNA extraction to digest many of the contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects nucleic acids from nuclease attacks.
What are the applications of Proteinase K?
Applications of Next Generation Sequencing (NGS) and Microarray Technology:
Nucleic acid purification by inactivating nucleases when extracting DNA and RNA from yeast, bacterial and mammalian cells and plant cell lysates;
Improve the cloning efficiency of PCR products;
Sample preparation for quantification of DNA adduct levels by accelerator mass spectrometry;
Inactivation of enzyme mixtures in ribonuclease protection assays;
Add to extraction procedures to optimize RNA yield from primary breast tumors for microarray studies and more
Applications of Proteinase K in Molecular Biology:
Detection of bovine spongiform encephalopathy proteins that are uniquely resistant to proteolytic degradation;
Tissue digestion (denaturation of proteins) as an alternative sample preparation method for quantitative analysis using liquid chromatography-tandem mass spectrometry;
Specialized modification of cell surface proteins to analyze membrane structure for protein localization;
Generate protein fragments for functional study characterization.
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