Author/Editor		Kuhelj, R; Dolinar, M; Pungerčar, J; Turk, V
Title		The preparation of catalytically active human cathepsin B from its precursor expressed in Escherichia coli in the form of inclusion bodies
Type		članek
Source		Eur J Biochem
Vol. and No.		Letnik 229, št. 2
Publication year		1995
Volume		str. 533-9
Language		eng
Abstract		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/l 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.
Descriptors		CATHEPSIN B AMINO ACID SEQUENCE BASE SEQUENCE CATHEPSIN B CLONING, MOLECULAR CYSTEINE DNA PRIMERS ESCHERICHIA COLI HYDROGEN-ION CONCENTRATION KINETICS MOLECULAR SEQUENCE DATA OXIDATION-REDUCTION PROTEIN FOLDING PROTEIN PRECURSORS PROTEIN PROCESSING, POST-TRANSLATIONAL RECOMBINANT PROTEINS