Proteinase K

Proteinase K is a broad-spectrum serine protease that was first isolated from the fungus Engyodontium album (formerly known as Tritirachium album). It is widely used in molecular biology to digest proteins and remove contamination from preparations of nucleic acid, thereby facilitating the study of DNA and RNA without interference from proteins.

Overview

Proteinase K cleaves peptide bonds at the carboxyl side of aliphatic and aromatic amino acids, making it highly effective in denaturing proteins, including those that are typically resistant to proteolytic cleavage. This enzyme is stable over a wide range of temperatures and pH levels, retaining activity in the presence of detergents such as sodium dodecyl sulfate (SDS) and urea, which makes it particularly useful in the isolation of nucleic acids from biological samples.

Applications

The primary application of Proteinase K is in the preparation of DNA and RNA for molecular biology experiments. It is used to remove protein contamination from nucleic acid extracts, a crucial step in processes such as PCR, restriction digestion, and cloning. Additionally, Proteinase K is employed in the removal of nucleases that can degrade DNA or RNA during extraction processes.

Mechanism of Action

Proteinase K works by cleaving the peptide bonds between amino acids in proteins. Its broad specificity allows it to target a wide range of proteins, effectively digesting them into smaller peptides and amino acids. This action helps to inactivate nucleases and other enzymes that could interfere with the analysis of nucleic acids.

Usage in Molecular Biology

In molecular biology, Proteinase K is often used in the extraction and purification of DNA and RNA. It is particularly useful in protocols requiring the removal of proteins from nucleic acid samples, such as genomic DNA extraction from animal tissue, plasmid DNA isolation, and RNA extraction from cells or tissues.

Preparation and Storage

Proteinase K is typically supplied in powder form and is reconstituted with water or buffer before use. The enzyme solution can be stored at -20°C for long-term use. However, repeated freeze-thaw cycles should be avoided to maintain its activity.

Safety and Handling

While Proteinase K is not considered highly hazardous, standard laboratory precautions should be observed when handling this enzyme. Protective clothing, gloves, and eye protection are recommended to prevent skin and eye contact.

Conclusion

Proteinase K is a versatile and indispensable tool in molecular biology, enabling the efficient preparation of nucleic acid samples free from protein contamination. Its broad specificity, stability under various conditions, and compatibility with detergents make it a valuable asset in the laboratory.