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Expression and purification of active human recombinant cathepsin K in E. coli
Author(s) -
Tha Soe,
McConnell Matthew,
Berryman Laura,
Chmielowski Jennifer,
Bordowitz Michael,
Liang Kenneth,
Lafollette Jessica,
Wen Lisa,
McConnell Rose
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.683.7
Subject(s) - recombinant dna , chemistry , microbiology and biotechnology , cathepsin , inclusion bodies , biochemistry , cathepsin h , enteropeptidase , cathepsin l , affinity chromatography , cathepsin b , fusion protein , enzyme , biology , gene
Cathepsin K, a lysosomal cysteine protease, is abundantly expressed in osteoclasts and involved in bone remodeling and resorption. It has been reported that inhibitors of cathepsin K show great potential in the treatment of osteoporosis. The objective of this research is to prepare functional recombinant cathepsin K enzyme which will be used in the development of new cathepsin K inhibitors. Human procathepsin K cDNA has been amplified by PCR and successfully subcloned into pET15b expression vector. Overexpression of the recombinant His‐tagged fusion protein in BL21(DE3)pLysS host cells was found mainly in inclusion bodies. The inclusion bodies were solubilized by 6 M guanidine hydrochloride and the recombinant procathepsin K protein purified using Ni‐NTA affinity column. The purified protein was refolded by dilution followed by dialysis. The procathepsin K protein has been autoactivated and its proteolytic activity assayed by monitoring the increase in absorbance at 405 nm due to hydrolysis of chromogenic substrate, Z‐Phe‐Arg‐pNA. The availability of the functional cathepsin K enzyme will permit a drug discovery program for development of newer effective therapy for the treatment of osteoporosis in the near future. Supported by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)