CHAPTER 16Proteinase K (EC 3.4.21.14)
Patricia J. Sweeney and John M. Walker
- Introduction
Proteinase K is a serine protease and the main proteolytic enzyme
produced by the fungus Tritirachium album Limber (1). The enzyme
has a broad 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 mam-
malian cell lysis. This allows the use of proteinase K in conjunction
with cell lysis resulting in the rapid degradation of released intracel-
lular 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 pro-
tein 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). The proteinase K method for extracting RNAis a well established
From: Methods in Molecular Biology, Vol. 16: Enzymes of Molecular Biology
Edited by: M. M. Burrell Copyright ©1993 Humana Press Inc., Totowa, NJ305