title: Efficient processing of procathepsin K to the mature form.
abstract: The proteolysis of collagen fibrils by cathepsin K is a hallmark of bone catabolism and tissue degeneration. The production of active recombinant cathepsin K is central for our ability to study the mechanisms by which these processes occur. Here we report an efficient processing method for the preparation of recombinant cathepsin K expressed in Pichia pastoris. Methanol precipitation of crude media and autoactivation in the absence of a reducing agent allows for the reversible inhibition of the enzyme prior to subsequent purification steps. The resultant purified enzyme is both resistant to autolysis and effective at cleaving collagen.
authors.affiliation: Genetics Unit, Shriners Hospital for Children and Department of Surgery, McGill University, Montreal, Quebec, Canada.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Emmott
authors.forename: John S
authors.initials: JS
authors.lastname: Mort
mesh_terms: D000595:Amino Acid Sequence; D056657:Cathepsin K / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024043:Collagen Type II / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D000432:Methanol / Q000737:chemistry; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D024043:Collagen Type II; D011994:Recombinant Proteins; C118659:procathepsin K; D056657:Cathepsin K; D000432:Methanol
keywords: Autoactivation; Cathepsin K; Cysteine protease; Pichia pastoris; Proenzyme processing; Protein expression