Proteinase K is a serine protease and the main proteolytic enzyme produced by the fungusTritirachium albumLimber (1 ). The enzyme has abroad specificity, cleaving peptide bonds C-terminal to a number of amino acids. The enzyme is produced, together with other proteases and an aminopeptidase, during stationary phase when the fungus is grown by submerged culture. The enzyme is so named because the organism can grow on native keratin as sole carbohydrate and nitrogen source owing to the enzyme’s ability to digest keratin. Because of its broad substrate specificity, high activity, and its ability to digest native proteins, proteinase K has found considerable use in procedures where the inactivation and degradation of proteins is required, particularly during the purification of nucleic acids. The enzyme retains activity in the presence of 0.5% sodium dodecyl sulfate, which is used in mammalian cell lysis. This allows the use of proteinase K in conjunction with cell lysis resulting in the rapid degradation of released intracellular nucleases and the subsequent isolation of intact nucleic acids. Following digestion, degraded protein is routinely removed by phenol extraction. For example, proteinase K has been used to degrade protein during the isolation of high-mol-wt eukaryotic DNA for cloning in phage or cosmid vectors (2 –4 ), to remove protein during plasmid (5 ) and lambda phage DNA (6 ) isolation, and to remove protein from protein DNA complexes produced during DNA footprinting analysis (7 ).
