Proteinase K Ultrapure (250 mg)

QIAGEN offers Proteinase K Ultrapure Grade, a recombinant subtilisin-related serine protease derived from Parengyodontium album (Tritirachium album) and expressed in Komagataella phaffii (Pichia pastoris) as a 28.9 kDa protein. It is widely used for protein digestion, including DNases and RNases, during nucleic acid preparations without compromising the integrity of isolated DNA or RNA.

On arrival the enzyme should be stored at –20°C. In its original unopened container it maintains activity ≥35 U/mg and specific activity ≥45 U/mg for at least 24 months, with 2.5-fold increased solubility and DNA content ≤0.1 pg/mg. It is highly soluble in water (≥50 mg/ml), with DNA content reported as ≤0.1 pg/mg from Escherichia coli and Komagataella phaffii. One unit is defined as the amount that hydrolyses urea-denatured hemoglobin to produce a colour equivalent of 1 µmol tyrosine per 1 minute at 37°C and pH 7.5 (Folin & Ciocalteu's method), where 1 U = 1 mAnsonU.

The enzyme is typically used at 50–200 µg/mL in nucleic acid preparations at pH 7.5–8.5 and 37–55°C, with incubation times from 30 minutes to 18 hours. It cleaves proteins preferentially behind hydrophobic amino acids and hydrolyses peptides down to a tetrapeptide. Working pH range is 4.0–12.0 with optimum pH 7.5–8.5; full activity is maintained over several hours at pH 6.5–10.0. Working temperature range is 20–65°C with optimum 50–56°C. Activity is stimulated by 0.2–1% SDS or 1–4 M urea, and Ca2+ (1–6 mM) provides prolonged stability against autolysis and increases thermal stability. The enzyme is not inactivated by chelating agents (e.g., EDTA), chaotropic salts, detergents, metal ions, thiol reagents, or trypsin-specific inhibitors. It is typically denatured by phenol extractions or inactivated by heating above 65°C or with inhibitors such as PMSF or DIFP.