Developing an enzyme which achieves the same application performance as liquid Proteinase K at the same protein concentration.
Proteinase K is a subtilisin-type serine endopeptidase isolated from Tritirachium album. This enzyme displays a broad substrate specificity which allows it to efficiently and effectively hydrolyse a wide range of proteins. Proteinase K is active over a wide pH and temperature range.
The extensive specificity towards protein makes it an ideal candidate for use in many molecular biology applications such as:
With the growing requirements for early detection of chronic diseases and identification of infectious diseases by genetic and molecular testing, the demand on the raw materials for these tests has increased exponentially. Proteinase K is one of the fundamental components of these tests responsible for removing unwanted proteins from nucleic acid preparations. Biocatalysts Ltd and BRAIN AG were aware of the growing demand for a cost-effective, high quality Proteinase K and developed a strategy to produce a Proteinase K alternative.
Proteocut K, is a broad spectrum serine protease that was tested by an external laboratory to confirm it meets the requirements needed for use in molecular biology applications. These tests prove Proteocut K is a high quality, cost effective alternative to Proteinase K.
Proteinase K is used in molecular biology applications where the customer requires that any replacement enzyme has an endopeptidase activity that performs at least as well as liquid Proteinase K in performance. This was a minimum requirement for developing Proteocut K and it was an also an important part of our strategy to consider cost in use to ensure that the enzyme developed was cost-effective as well as a high-quality Proteinase K alternative. This resulted in the production of a serine endopeptidase that has an activity ≥1,450 U/ml.
Initially, we identified a suitable subtilisin protease which displayed a broad substrate specificity, that worked well over a broad pH and temperature range and was produced at an acceptable yield.
BRAIN AG then developed a lab-scale downstream protein purification (DSP) process, to deliver enhanced activity and increased performance of this enzyme. This DSP was transferred to Biocatalysts Ltd for scaling up to allow us to manufacture commercial quantities of this enzyme with high purity, high activity, and good stability. To ensure that the enzyme had the correct activity level, and that we were comparing like for like Proteocut K with Proteinase K, we assayed the product with the Haemoglobin activity assay* – the recognised standard used to measure the activity of Proteinase K. This introduced a new assay to Biocatalysts Ltd which the enzyme assay team put through our full assay validation process to create a reproducible and robust assay that could be used to compare the activity of our enzyme to other commercially available Proteinase K products.
This product was then sent to an Independent External Laboratory to test its performance against a commercially available liquid Proteinase K in different molecular biology application trials.
|Application Tests||Result Proteocut K (PK909L)||Result Proteinase K|
|Hydrolysis of 5ug Bovine Serum Albumin by 1µg Proteocut K (PK909L) or Proteinase K||>99% Hydrolysis with/without 2% SDS at 37°C in 2 hours||Proteolysis greatly reduced in the presence of SDS|
|Hydrolysis of 16mg Bacterial Cell Lysate by 1µg Proteocut K (PK909L) or Proteinase K||≥90% Hydrolysis with/without 2% SDS at 37°C in 2 hours||≥90% Hydrolysis with/without 2% SDS at 37°C in 2 hours|
|Hydrolysis of 20mg CHs5 Fish Cell Lysate by 1µg Proteocut K (PK909L) or Proteinase K||≥95% Hydrolysis with/without 2% SDS at 37°C in 2 hours||<90% Hydrolysis with/without 2% SDS at 37°C in 2 hours|
|Plasmid DNA Extraction||Result Proteocut K (PK909L)||Result Proteinase K|
|Biotek E.Z.N.A plasmid DNA mini kit used to isolate plasmid DNA from 5ml culture of E.coli DH5α containing a plasmid. 10µl of Proteocut K (PK909L) or 10µl of Proteinase K were added in place of kit protease during DNA isolation.||40.6 ng/µl of high-quality plasmid DNA isolated after short incubation time (3-5 minutes) as measured via nanodrop and gel electrophoresis.||37.7 ng/µl good quality plasmid DNA isolated after short incubation time (3-5 minutes) as measured via nanodrop and gel electrophoresis.|
|Deactivation Test||Result Proteocut K (PK909L)||Result Proteinase K|
|500ng Proteocut K (PK909L) or Proteinase K incubated at 80ºC for 6 mins||100% Inactivation as shown with AbCam* assay||Residual activity detected with AbCam* assay|
*One unit of Proteinase K hydrolyses urea-denaturated haemoglobin producing colour equivalent of 1 μmol tyrosine per 1 min at 37°C and pH = 7.5 (Folin & Ciocalteu’s method).
Biocatalysts Ltd have scaled up production of Proteocut K, a cost-effective alternative to liquid Proteinase K that is suitable to use in molecular biology applications. During this process Biocatalysts Ltd successfully replicated and validated the Haemoglobin activity assay to allow us to develop Proteocut K, which has an activity level of ≥1,450U/ml. We worked closely with an Independent Laboratory (with expertise in Proteinase K) who designed and executed various molecular biology application trials which confirmed our product performed as well as liquid Proteinase K.