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Overexpression of recombinant human procathepsin D in E. coli
Author(s) -
Keefer Ryan,
McConnell Matthew,
Ye Alice,
Chmielowski Jennifer,
Berryman Laura,
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.683.4
Subject(s) - recombinant dna , cathepsin d , microbiology and biotechnology , xhoi , cathepsin , fusion protein , chemistry , cathepsin l1 , enzyme , biology , biochemistry , gene , bamhi
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)