Open AccessThe Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in Escherichia coli in the Form of Inclusion Bodies
Author(s) -
Kuhelj Robert,
Dolinar Marko,
Pungerčar Jože,
Turk Vito
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.0533k.x
Subject(s) - escherichia coli , inclusion bodies , biochemistry , enzyme , chemistry , cathepsin b , recombinant dna , cathepsin l , complementary dna , amino acid , pepsin , cathepsin , active site , microbiology and biotechnology , biology , gene
A cDNA clone encoding human procathepsin B was expressed at a high level in Escherichia coli using a T7 polymerase expression system, resulting in the formation of insoluble cytoplasmic protein aggregates (inclusion bodies). The recombinant product was solubilized and renatured by refolding and reoxidation. The proenzyme was subsequently processed with pepsin to produce an enzymically active enzyme. By systematic variation of the parameters influencing the folding, formation of disulphide bonds, and processing of procathepsin B to the catalytically active mature form, a simple renaturation procedure was designed, allowing the production of about 3 mg purified active cathepsin B/1 E. coli culture broth. The enzyme obtained in this way consists of a single chain and, as a consequence of pepsin treatment, possesses a three‐amino‐acid extension at its N‐terminus. The enzyme has similar kinetic and immunological properties to native human cathepsin B.