Article

The function of proteinase K in DNA extraction for molecular diagnostics

Proteinase K for sample preparation – why, what and where?
cb sample preparation
What is proteinase K and how does it work?

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.

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What is the purpose of proteinase K in molecular diagnostics?

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 (Table 1).

Table 1: Benefits of proteinase K for sample preparation prior to DNA isolation

Broad spectrum of activity

Inactivates and degrades proteins– enabling rapid degradation of sample derived nucleases.

Disrupts cell membranes2 – efficiently releasing nuclear material.

Neutralizes nucleases – preventing DNA and RNA degradation.3

Digests contaminant proteins3 – that inhibit reverse transcriptases and polymerases, or disrupt primer binding.

Active under harsh conditions

Broad temperature range – between 25 and 60oC.4

pH stable – between 4.0 and 12.5,4 active between pH 7.5 and 12.0,5 with optimum activity pH of 8.0,4 which broadens the applications for which it is useful.

Denaturants – stimulated by denaturants (SDS and urea),6 so these can be used to fine-tune reaction rates.

Detergents – remains active even in the presence of detergents used in lysis buffers.7

Unique functional characteristics

Deactivated above 95 oC 8 – preventing interference with high-temperature PCR cycling protocols.

Active but thermo-unstable in the absence of Ca2+ 9 – allowing use of calcium salts and heat to control activity.

Sourcing proteinase K for molecular diagnostics

The performance characteristics and stability of proteinase K make it ideal for processing biological samples for molecular diagnostic assays, but other factors such as batch-to-batch predictability, formulation, stability and availability are just as important when designing kits and protocols.

predicatability

Predictability

Knowing exactly how a given batch of proteinase K will perform, and under what conditions, is essential. Proteinase K was originally isolated from the mold Tritirachium album,6 and has been widely studied and well characterized. However, while some manufacturers still extract proteinase K from its native source, this can lead to batch-to-batch variability, requiring laborious characterization of each new batch to ensure consistent performance. As a result, it is now widely produced as a recombinant enzyme, but there will still be some variation between sources. 

Purity is also an essential consideration to help guarantee performance and compatibility with other components used in sample preparation and/or diagnostic steps. Look for a supplier that guarantees the specific activity and high purity in proteinase K preparations intended for sample processing, as this will help to ensure efficient extraction of nuclear material, optimization of downstream diagnostic assays, and accuracy of results.

lyo ready

Formulation

Proteinase K is available in a variety of formulations – both in solution and lyophilized – to provide flexibility for a broad range of applications: where one protocol might require a solution in a centrifuge tube, another might need powder-coated beads. 

stability

Stability

When remote, point-of-care testing is needed, samples must often be taken and processed on site, away from the facilities available in a central clinical laboratory. Proteinase K has excellent long-term stability at room temperature, meaning that it can be shipped and stored long-term without refrigeration. This increases the potential to process samples in remote, inaccessible areas and developing countries, where costs can be a barrier to healthcare.

predicatability

Availability

When designing, developing and scaling up to manufacture, security of reagent supplies is an important factor to consider when choosing to use one source over another. Changing suppliers can compromise product consistency and quality, and might mean that a kit or protocol design needs reoptimizing, which costs time, money and customer goodwill. A reliable supplier with an excellent track-record of expertise and stock provision can therefore make all the difference.

Summary

Proteinase K is a vital tool for the digestion of proteins in biological samples prior to DNA isolation for molecular diagnostics. It is therefore important to consider its source and formulation when developing a new assay, ensuring the chosen product has the functionality and stability needed. Choosing the best quality proteinase K from a reliable supplier helps to guarantee a reliable and efficient assay, simplifying molecular diagnostic workflows and increasing user satisfaction.

Do you need proteinase K for your next project?

Roche CustomBiotech has been producing high quality, high purity proteinase K for over two decades. The company’s high capacity production plant has a strong track record of responding quickly to unpredictable rises in demand.11

Roche CustomBiotech’s Proteinase K is:

  • Recombinant and identical to the native enzyme – ensuring outstanding reliability and purity, and meeting all the requirements of diagnostic assay manufacturers. 6,10

  • PCR grade quality – guaranteed to have a low DNA content and be free from contaminant nucleases.6,10

  • Available as both a high concentration solution and a lyophilizate – to suit various applications.6,10

  • Stable for 18 months at 2–8 oC, and for 1 year at 25 oC, enabling manufacturing flexibility and long shelf life of final products.6,10

Reach out to your regional Roche CustomBiotech representative for more information on proteinase K.
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Regulatory disclaimer: For further processing only.

References:

  1. Sweeney PJ, Walker JM. Proteinase K (EC 3.4.21.14). Methods Mol Biol. 1993;16:305-311. doi:10.1385/0-89603-234-5:305
  2. Islam MS, Aryasomayajula A, Selvaganapathy PR. A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines (Basel). 2017;8(3). doi:10.3390/MI8030083
  3. Mótyán JA, Tóth F, Tőzsér J. Research Applications of Proteolytic Enzymes in Molecular Biology. Biomolecules. 2013;3(4):923. doi:10.3390/BIOM3040923
  4. Farrell RE. Resilient Ribonucleases. RNA Methodologies. 2010:155-172. doi:10.1016/B978-0-12-374727-3.00007-3
  5. Ebeling W, et al. Proteinase K from Tritirachium album Limber. Eur J Biochem. 1974;47(1):91-97. doi:10.1111/J.1432-1033.1974.TB03671.X
  6. Roche CustomBiotech. 2024. Proteinase K, rec., PCR Grade, lyo. https://custombiotech.roche.com/global/en/products/cb/proteinase-k-recombinant-pcr-grade-lyophilizate-3614817.html [Accessed May 1, 2024]
  7. Saenger W. Proteinase K. Handbook of Proteolytic Enzymes. 2013;3:3240-3242. doi:10.1016/B978-0-12-382219-2.00714-6
  8. Goldenberger D, et al. A simple "universal" DNA extraction procedure using SDS and proteinase K is compatible with direct PCR amplification. PCR Methods Appl. 1995;4(6):368-370. doi:10.1101/gr.4.6.368
  9. Bajorath J, Hinrishs W, Saenger W. The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem. 1988;176(2):441-447. doi:10.1111/J.1432-1033.1988.TB14301.X
  10. Roche CustomBiotech. 2024. Proteinase K, rec., PCR Grade, sol. https://custombiotech.roche.com/global/en/products/cb/proteinase-k-recombinant-pcr-grade-from-titrirachium-album-expressed-in-pichia-pastoris-solution-3614816.html [Accessed May 1, 2024]
  11. Roche CustomBiotech. 2024. A legacy of excellence: 20 years of Proteinase K production in Penzberg. https://custombiotech.roche.com/global/en/post-listing/2023/a-legacy-of-excellence--20-years-of-proteinase-k-production-in-p.html [Accessed May 1, 2024]
custombiotech

Roche CustomBiotech is renowned for delivering high-quality reagents that meet stringent industry requirements. 

Why proteinase K – Proteinase K’s non-specific action, unique functional characteristics, and activity and stability under harsh conditions make it ideal for pre-processing samples for molecular diagnostic assays.

What to look for – When sourcing proteinase K for industrial use, e.g. in diagnostic tests, consider whether the batch you’re buying has a specified activity, is of high purity, comes in different formulations, and has a secure supply chain.

Where to find it – Roche CustomBiotech has proven market experience, with security of supply.