No abstract

Cathepsin D is an aspartyl lysosomal protease that catalyzes protein cleavage. It is involved in the process of tumor invasion and metastasis. The cathepsin D enzyme has been considered as a potential target for cancer therapy. The objective of this research is to prepare functional recombinant cathepsin D enzyme which will be used in the development of new cathepsin D inhibitors. Human procathepsin D cDNA, purchased from OpenBiosystems, was amplified by PCR and sub‐cloned into XhoI site of pET15b. The procathepsin D‐pET15b construct was then transformed into JM109. The recombinant plasmid was isolated from the JM109 and transformed into BL21(DE3)pLysS cells for expression. Expression of recombinant procathepsin D protein in the transformed BL21(DE3)pLysS cells was induced with IPTG. The level of expression was analyzed by SDS‐PAGE. High level expression (greater than 50% of total cellular proteins) of the His‐tagged procathepsin D fusion protein was achieved when cells were grown in terrific broth. The ability to produce procathepsin D enzyme will permit a drug discovery program for development of newer effective inhibitors of cathepsin D in the near future.Supported by National Cancer Institute at NIH Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)

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)