The Preparation of Catalytically Active Human Cathepsin B from Its Precursor Expressed in Escherichia coli in the Form of Inclusion Bodies

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 BA 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.Keywords. Cathepsin B ; renaturation ; refolding ; processing ; expression.Among the lysosomal cysteine proteases, cathepsin B is the most abundant and the most thoroughly studied. This enzyme takes part in many normal cellular processes, including intracellular protein degradation (Barrett et al., 1988), bone resorption (DelaissC and Vaes, 1992) and antigen processing (Guagliardi et al., 1990). Several pathophysiological states have also been attributed to its action, e.g. Alzheimer's disease (Cataldo and Nixon, 1990), arthritic processes (Van Norden et al., 1988), cancer invasion and metastasis (Sloane et al., 1994), and others. In vivo, the protein is synthesized as a preproenzyme. After being glycosylated, procathepsin B is targeted to the lysosomes by the mannose 6-phosphate signal. Presumably in endosomallprelysosomal compartments it is converted by limited proteolysis into the single-chain form of the mature enzyme, which can be further processed in the lysosomes in a cell-type-specific manner into two-chain cathepsin B consisting of a heavy and light chain linked together by a disulphide bond (Hanewinkel et al., 1987;Mach et al., 1992).In vitro proteolytic processing of recombinant rat procathepsin B, expressed in yeast, has been studied by Rowan et al. (1992). These authors showed that cathepsins D, L, mature cathepsin B itself, and pepsin can produce a processed single-chain form of cathepsin B from its precursor. N-terminal sequencing revealed that these proteins are all elongated by a few residues with respect to the mature form found in vivo. Mach et al. (1994) showed that human procathepsin B can be autocatalytically acti- vated and processed, the mechanism of this process probably being unimolecular.The X-ray crystal structure of human liver cathepsin B has already been determined (Musil et al., 1991); this protein is the first mammalian cysteine protease whose tertiary structure has been determined.In addition to its well characterized endopeptidase activity, cathepsin B also exhibits exopeptidase (dipeptidyl carboxypeptidase) activity (Takahashi et al., ...

Cathepsin B in infiltrated lymph nodes is of prognostic significance for patients with nonsmall cell lung carcinoma

“…The color was developed by adding 3,3Ј-diaminobenzidine tetrahydrochloide (0.3 mg/mL in 0.05 M Tris buffer, pH 7.5), including 0.2% hydrogen peroxide for 10 -15 minutes or by using the Vector Red Substrate Kit according to the instructions of the respective manufacturer. The following control assays were performed: 1) preadsorption of the antigen (recombinant full-length cath B, prepared by Kuhelj et al 31 ) with the antibody (rab- bit anti-human cath B IgG) in a one-to threefold molar ratio; 2) incubation without primary antibody; and 3) incubation without the secondary antibody. Tissue sections were evaluated by the pathologist (K.K.…”

Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli

“…The cysteine proteinase activity assay is best monitored by synthetic fluorogenic substrates such as aminomethylcoumarin (AMC). The cathepsins with endopeptidase activity can be followed by their Z-Phe-Arg-AMC cleaving activity (e.g., cathepsins B, F, K, L, O, S and V;Brömme 2001 Hwang and Chung (2002) Cathepsin B pH 3.5, porcine pepsin (1/100) Z-Phe-Arg-AMC, Z-Arg-Arg-AMC Kuhelj et al (1995) Cathepsin C pH 4.5, 20 mM citric acid, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, cathepsin L and S Gly-Phe-pNA, Gly-Arg-pNA Dahl et al (2001) Falcipain-2 pH 5.5, 100 mM sodium acetate, Cysteine protease inhibitors provide a useful tool to study protease activity. Compounds synthesized included a wide range of peptide aldehydes, methyl nitriles and ketones as reversibly acting inhibitors and halomethyl ketones, diazomethanes, acyloxymethyl ketones, O-acylhydroxamates, and epoxysuccinyl derivatives as irreversible inhibitors (Lecaille et al 2002).…”

“…Proteins have been purified based on (1) their surface properties, i.e., ion exchange chromatography (IEC); (2) their net charge, i.e., size exclusion chromatography (SEC); (3) their high-affinity ligands, i.e., immobilized metal affinity chromatography (IMAC; Brömme 2001). IEC has been widely used to purification recombinant papain-like cysteine protease (Table 1), such as cathepsin B (Kuhelj et al 1995), Falcipain-2 , vinckepain-2 (Singh et al 2002), cathepsin F-like cysteine protease domain (Miyaji et al 2010) and cruzipain (Eakin et al 1992). Using IEC column as a refolding reactor, purification and refolding can be achieved simultaneously.…”

“…The E. coli strain BL21(DE3) are commonly used as expression hosts and are grown in Luria-Bertani medium contains 50-100 lg/ml ampicillin at 37°C until OD 600 0.6-0.8. The T7 RNA polymerase inducible system is widely used for the expression of papain-like cysteine proteases, such as cathepsin O (Velasco et al 1994), cathepsin B (Chen and Sun 2012; Kuhelj et al 1995), cathepsin L Lee et al 2012), cathepsin S (Kim et al 2010;Kopitar et al 1996;Tobbell et al 2002) and cathepsin F (Santamaria et al 1999). Protein expression is induced by addition of isopropyl-b-D-thiogalactoside (IPTG) at a final concentration variable from 0.1 to 2 mM for 3-5 h. However, most of the papain-like cysteine proteases are expressed in inclusion bodies.…”

The steroid hormone 20-hydroxyecdysone upregulated the protein phosphatase 6 for the programmed cell death in the insect midgut

“…Then, the washed inclusion bodies were dissolved in buffer C (0.1 M Tris-HCl, pH 8.0, 10 mM dithiothreitol, 8 M urea). The proteins were then refolded in dialysis buffer (0.1 M Tris-HCl, pH 8.0, 5 mM EDTA, 5 mM cysteine) for 16 h at 4°C (Kuhelj et al 1995). The refolded samples were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the target protein was excised from the gel.…”