Proteinase K: Preparation and Function

Proteinase K is a proteolytic enzyme (a serine protease) that is purified from the mold Tritirachium album. In solution, it is stable over a pH range 4.0–12.5 with an optimum of pH 8.0, and a temperature range 25–65°. Proteinase K is prepared commonly as a 20 mg/ml stock solution in sterile water (stable for 1 year at −20°) or in a solution of 50 mM Tris, pH 8.0, 1 mM CaCl2 (stable for months at 4°). It is generally used at a working concentration of up to 50 μg/ml in any of a number of buffer formulations, including those that contain up to 0.5% SDS^[1].

Preparation

You can prepare Proteinase K solution by following these steps:

1. Weigh out the desired amount of Proteinase K powder using a balance. The amount will depend on the specific protocol, but commonly used concentrations range from 10 to 100 mg/mL.

2. Add the Proteinase K powder to a tube or container.

3. Add an appropriate volume of buffer or water to the tube or container. The specific buffer or solvent used will depend on the application and protocol being followed. Commonly used buffers include Tris-HCl, ethylene diamine tetraacetic acid (EDTA), or TE buffer.

4. Mix the contents of the tube or container well by vortexing or pipetting up and down.

5. Incubate the tube or container at the recommended temperature (most used at 37 °C) and time for the specific protocol being followed.

6. After incubation, the Proteinase K solution is ready to use for downstream applications.

Function

Proteinase K is a broad-spectrum serine protease that cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. It is capable of digesting a wide variety of proteins, including those that are resistant to other proteases, making it a useful tool across biochemistry and molecular biology.

The reliability and sensitivity of any molecular diagnostic test requires that the reverse transcription (if needed) and DNA amplification phases are efficient, accurate and reliable. However, if the biological sample being tested has been inadequately prepared, these vital processes can be significantly impacted, which compromises the integrity and reliability of any test result.

Samples for molecular testing often need some degree of pre-processing before analysis, typically involving the digestion of structural proteins, inactivation of nucleases, and RNA or DNA isolation. This is where Proteinase K plays a vital role, with its ability to break down the protein components of the cell membrane to allow access to the genetic material, and to remove nucleases that could degrade DNA and RNA. It is commonly used in sample pre-processing due to its distinctive activity profile, ability to work under harsh conditions and unique functional characteristics.

Reference

[1] Farrell, R. (2010). Chapter 7 – Resilient Ribonucleases. 0. https://doi.org/10.1016/B978-0-12-374727-3.00007-3