title: Folding and activation of human procathepsin S from inclusion bodies produced in Escherichia coli.
abstract: Human procathepsin S was produced in the form of insoluble inclusion bodies in Escherichia coli using an inducible T7-based expression system. After cell disruption, the dissolved inclusion body proteins were S-sulphonated with 2-nitro-5-thiosulphobenzoate and purified by gel filtration. Recombinant procathepsin S was renatured at pH 7.6 by a two-step dilution which significantly increased the yield of production compared to single-step dilution. The proenzyme was autocatalytically processed to active cathepsin S at pH 4.5 in the presence of an excess of cysteine and catalytic amounts of dextran sulphate. Most of the loss of procathepsin S occurred during folding, probably because of aggregation. Concentrations lower than 20 microgram/ml of procathepsin S were necessary to minimise such aggregation. The recombinant cathepsin S was catalytically active on fluorogenic substrates and had kinetic properties similar to those of recombinant enzyme produced in yeast. The expression, renaturation, and activation procedures used enable the production of up to 2 mg of catalytically active recombinant human cathepsin S/l fermentation broth.
authors.affiliation: Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Ljubljana, Slovenia.
authors.forename: G
authors.initials: G
authors.lastname: Kopitar
authors.forename: M
authors.initials: M
authors.lastname: Dolinar
authors.forename: B
authors.initials: B
authors.lastname: Strukelj
authors.forename: J
authors.initials: J
authors.lastname: Pungercar
authors.forename: V
authors.initials: V
authors.lastname: Turk
mesh_terms: D001483:Base Sequence; D002403:Cathepsins / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry*; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D002403:Cathepsins; C028217:cathepsin S