SMU Medical Journal

Arumugam Jayakumar ¹, Hua-Kang Wu ¹, Katrina Briggs ¹, Thomas Shellenberger ¹, Ya'an Kang ¹, Kenji Mitsudo ¹, Mary Wang ¹, Mitchell J. Frederick ¹, Ying Henderson ¹, Paula R. Holton ¹, Venugopal Radjendirane ², Karthik Jayakumar ³, Dieter Brömme ⁴

Affiliations
1 Department of Head and Neck Surgery, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, US
2 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030, US
3 The American University of Antigua, Mount Sinai School of Medicine, New York, New York 10029-6574, US
4 Department of Human Genetics Mount Sinai School of Medicine, New York, New York 10029-6574, US

Abstract

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.

Keywords

Cathepsink, Sf9, Metal Affinity Chromatography, HNSCC, Headpin.

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