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Radjendirane, Venugopal
- Expression, Purification and Characterization of Hexahistidine- Tagged Human Cathepsin K in High Five Cells
Abstract Views :252 |
PDF Views:153
Authors
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
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
Source
SMU Medical Journal, Vol 2, No 1 (2015), Pagination: 1-18Abstract
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.- Current Status of LEKTI, a Physiological Inhibitor of Multiple Proteinases in the Skin-A Review
Abstract Views :204 |
PDF Views:137
Authors
Affiliations
1 Department of Experimental Therapeutics, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77054, US
1 Department of Experimental Therapeutics, The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77054, US