Expression, Purification and Characterization of Hexahistidine- Tagged Human Cathepsin K in High Five Cells

Cathepsin K is a protease with high collagenolytic and elastinolytic activity. A full-length cDNA clone of human cathepsin K was used to construct an expression system in insect cells. The recombinant protein had a C-terminal tag of six histidine residues, which allowed purification of this protein by a one-step Co²⁺-Sepharose affinity chromatography. Minimal amounts of pro-cathepsin K were secreted into the medium but most of the cathepsin K was present within infected cells. Very little processing of pro-enzyme to mature form occurred in High Five cells. Spontaneous in vitro activation of pro-cathepsin K to mature-cathepsin K occurred at pH 4.0. The ∼29 kDa mature-cathepsin K efficiently hydrolyzed the fluorogenic peptide substrate and r-headpin, a serine protease inhibitor down-regulated in head and neck cancer cell lines and tumor tissues, potently inhibited the r-catK activity in vitro. Taking cathepsin K as an example, we present a strategy, which should facilitate the expression of perhaps other cathepsins or proteases in general in insect cells.