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 pg/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/1 fermentation broth.Keywords: cathepsin S ; Escherichia coli ; inclusion bodies ; proenzyme ; renaturation ; processing.Cysteine proteinases, including cathepsin S, are capable of degrading extracellular matrix in various diseases (Sloane et al., 1990;Lah et al., 1992; GabrijeltiE et al., 1992). The degradation is thought to occur in the extracellular environment where the pH is neutral; among cysteine proteinases, only cathepsin S is active at this pH. The highest expression of human cathepsin S mRNA was found in lungs, heart muscle, and spleen, with a significantly lower expression in other tissues including brain (Shi et al., 1994), where the expression of rat cathepsin S is high (Petanceska and Devi, 1992). Very recently, it has been reported that cathepsin S may play a role in pathogenesis of the cerebral lesions of Alzheimer's disease or represent a response to their formation. It was suggested that cathepsin S could be partly involved in processing p-amyloid precursor protein to amyloid-p in the amyloid plaques (Lemere et al., 1995).Cathepsin S is a lysosomal cysteine proteinase (Turk and Bode, 1993). In contrast to other cathepsins, cathepsin S is present exclusively as a single-chain proteinase (Ritonja et al., 1991 ;Wiederanders et al., 1991) and is not glycosylated, although it contains a potential glycosylation site on its propeptide (Shi et al., 1992;Wiederanders et al., 1992). In contrast to cathepsins B and L, cathepsin S is both stable and active at neutral pH. It has a high endopeptidase activity against different proteins including elastin and collagen. Its substrate specificity shows some similarities with cathepsin L, but it differs distinctly in its S, subsite specificity (Xin et al., 1992).Human cathepsin S cDNA encodes a preproprotein of 331 amino acid residues, whereas the mature enzyme contains 217 residues (Shi et al., 1992;Wiederanders et al., 1992).The cDNA for human preprocathepsin S h...

Functional Expression of Dipeptidyl Peptidase I (Cathepsin C) of the Oriental Blood Fluke Schistosoma japonicum in Trichoplusia ni Insect Cells

“…Eluted fractions were analyzed by Coomassie-stained SDS-PAGE gels and by immunoblots probed with a monoclonal antibody (Qiagen) specific for the polyhistidine tag (RGSHHHH) fused to the NH 2 -terminus of the recombinant protein (not shown). The purified recombinant protein did not appear to be enzymatically active, even after repeated attempts at refolding in native buffers and/or activation with pepsin, as described by other workers (16,17). Notwithstanding the inactivity of the E. coli-expressed enzyme, it was used as the antigen to produce a polyclonal antiserum to S. japonicum DPP I.…”

Optimizing dentin bond durability: Control of collagen degradation by matrix metalloproteinases and cysteine cathepsins

“…[123]) and stabilization [124] of cathepsin (and possibly MMPs [116]), allows functional activity of cathepsins even in neutral pH. GAGs are able to accelerate the conversion of zymogen forms of cysteine cathepsins into mature forms at neutral pH, activating cathepsin B [123], cathepsin L [125], cathepsin S [106,126,127] and congopain [128]. The presence of GAGs in the dentin [129] and their release during acid-demineralization and subsequent proteolytic degradation of acid-demineralized dentin matrix [130] supports the possibility that GAGs are involved in in vivo processing of cysteine cathepsins in dentin.…”

Inhibition of Trypanosomal Cysteine Proteinases by Their Propeptides

“…The propeptide of mammalian and vegetal cysteine proteinases takes part in the proper folding, intracellular trafficking, or secretion of the mature proteinase, and in the control of proteolytic activity by blocking the active site (17)(18)(19). Isolated propeptides may still bind and inhibit their mature enzyme even when released from the proenzyme (20, 21).…”