Proteinase K is a serine protease used in molecular biology and biochemistry applications. These applications rely on Proteinase K’s proteolytic activity and its broad substrate specificity.
Proteolytic activity is an enzyme’s ability to break down proteins into peptides or amino acids.
Substrate specificity is an enzyme’s ability to recognize and act on a particular molecule (substrate).
The primary role of Proteinase K is to digest proteins in a sample without breaking down other molecules, such as DNA or RNA.
This article explains how the biochemical properties, mechanisms, and applications of Proteinase K help optimize experimental workflows.
Proteinase K is isolated from the fungus Tritirachium album. It is stable over a pH range of 4-12 and a temperature range of 37–60°C. The active site of this enzyme contains a set of the following three key amino acids known as the catalytic triad:
This enzyme can degrade resilient proteins such as keratin and collagen. As a result, it is widely used to prepare samples, particularly to extract nucleic acids from complex biological materials. It maintains its activity in the presence of detergents, chelating agents, and organic solvents. Therefore, it can be used in experiments with harsh conditions.
The catalytic triad of Proteinase K—serine, histidine, and aspartate—works together to cleave peptide bonds. Histidine abstracts a proton from serine’s hydroxyl group, leaving serine’s oxygen atom negatively charged (O⁻), making it a nucleophile. The activated serine then attacks the carbonyl carbon of the peptide bond. Histidine then holds the proton it abstracted from serine, giving it a temporary positive charge. Aspartate stabilizes this positive charge, ensuring that the reaction proceeds efficiently.
This coordinated activity of the catalytic triad breaks peptide bonds at hydrophobic, aliphatic, and aromatic amino acids, allowing Proteinase K to degrade proteins effectively.
Proteinase K digests contaminants during nucleic acid extraction, which ensures high yield and purity.
Proteins can unfold under high temperatures, strong acids or bases, detergents (such as SDS), urea, or other denaturing conditions. However, Proteinase K remains active under these conditions. It efficiently breaks down proteins to prevent contamination of the nucleic acids.
Recombinant protein research uses Proteinase K to remove host-cell proteins. This serine protease also helps in processing complex tissue samples.
Example:
Non-target proteins can cause interference in functional assays or antibody binding studies. Therefore, researchers use Proteinase K to prevent this interference when working with Stanniocalcin-1 Recombinant Protein.
Proteinase K is used in recombinant antibody technology to remove contaminating proteins. It is used to digest fusion tags after protein expression. This results in improved quality and specificity of recombinant antibodies.
Techniques like in situ hybridization and immunohistochemistry use Proteinase K for tissue digestion. It can permeabilize tissues without causing any damage to target structures.
It is active at temperatures around 50–60°C and a pH near 8.0. However, the conditions can be adjusted depending on the substrate and experimental requirements.
Inhibitors such as PMSF should only be used when intentional inhibition is required, as these serine protease inhibitors can completely abolish activity.
High substrate concentration may require higher enzyme amounts to achieve complete digestion, especially in complex tissue or protein mixtures.
Proteinase K can generate peptide fragments for mass spectrometry, which improves protein identification and characterization in complex biological samples.
The combination of Proteinase K digestion and recombinant antibody technology aids in:
Proteinase K is used in experiments involving Stanniocalcin-1 recombinant protein to help define interaction networks by selectively digesting unbound proteins. This allows for cleaner detection of physiologically relevant interactions.
As it is a potent protease, appropriate laboratory precautions are essential to prevent accidental protein degradation or skin exposure. It is typically supplied as a lyophilized powder or in a stabilizing buffer. It should be stored at -20°C to maintain activity.
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