Active papain renatured and processed from insoluble recombinant propapain expressed in <i>Escherichia coli</i>

Taylor, Mark A.; Pratt, Kathryn A.; Revell, Dean F.; Baker, Kenneth C.; Sumner, Ian G.; Goodenough, Peter W.

doi:10.1093/protein/5.5.455

Cited by 51 publications

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“…Shinde and Inouye (1993) proposed that propeptides are indispensible for proper protein folding, suggesting that they could play a role similar to that of chaperones. The fact that cathepsin L and papain, also members of the cysteine protease group, were expressed in the form of proenzymes (Smith and Gottesman, 1989;Taylor et al, 1992), further encouraged us in our decision to express cathepsin B as a proenzyme.…”

Section: Discussionmentioning

confidence: 99%

“…In this way, the T7 RNA polymerase basal activity was reduced, thus allowing cells to proliferate to high density before expression of procathepsin B was induced by IPTG. The use of the T7 lysozyme coding plasmid pLysS to prevent recombinant protein expression before induction was also obligatory in the case of propapain (Taylor et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

“…Since papain, another cysteine protease, has been produced in E. coli (Taylor et al, 1992), we also decided to attempt to produce cathepsin B using this system. The probability that polypeptide chains pos- sessing many cysteine residues would fold properly within the reducing milieu of E. coli cytoplasm is relatively low and insoluble inclusion bodies usually result, which was also the case in our endeavour to express procathepsin B.…”

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

Kuhelj¹,

Dolinar²,

Pungerčar³

et al. 1995

Eur J Biochem

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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., ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Kuhelj¹,

Dolinar²,

Pungerčar³

et al. 1995

Eur J Biochem

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“…Recombinant Fc⑀3-4⌬C was expressed in the E. coli host strain BL21 (DE3). Inclusion body protein was extracted from cell pellets using a procedure adapted from Bohmann and Tjian (18) and refolded according to the method of Taylor et al (19). We have previously described in detail the production and purification of an IgE Fc domain using these methods (7).…”

Section: Methodsmentioning

confidence: 99%

Disulfide Linkage Controls the Affinity and Stoichiometry of IgE Fcϵ3–4 Binding to FcϵRI

Hunt

Beavil

Calvert

et al. 2005

Journal of Biological Chemistry

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؊1 , similar to that determined previously for an isolated and partially folded C⑀3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two C⑀3 domains. Recent structural data indicate that conformational change in IgE is required to allow both C⑀3 domains to bind, and thus an allosteric inhibitor that prevents access to the second C⑀3 has the potential to reduce the ability of IgE to sensitize allergic effector cells. Immunoglobulin E (IgE)1 is the antibody class that plays a central role in the allergic response (1). Mast cells and basophils express a high-affinity receptor for IgE, Fc⑀RI, and it is the cross-linking of receptor-bound IgE on these cells by multivalent allergens that triggers the immediate release of preformed inflammatory mediators. The affinity of IgE for Fc⑀RI is uniquely high among Ig-receptor complexes, two to five orders of magnitude higher than that of IgG for its receptors Fc␥ RI-III (2). This is due principally to a very slow off-rate. The half-life of IgE bound to Fc⑀RI is hours (3) compared with only minutes for IgG bound to its receptors (4). Inhibition of IgE binding to Fc⑀RI or at least modulation of its binding kinetics is a potential therapeutic strategy.IgE Fc binds to the receptor with a 1:1 stoichiometry (5) using both Fc heavy chains (6). The crystal structure of the complex between the IgE fragment, Fc⑀3-4 (a homodimer of ⑀-chains consisting of the C⑀3 and C⑀4 domains), and a soluble fragment of the IgE-binding ␣-chain of the receptor, sFc⑀RI␣ (6), revealed the precise details of the involvement of the two C⑀3 domains in the complex. The extensive binding interface consists of two subsites, one contributed by each C⑀3 domain, and thus the maintenance of the dimeric site might be expected to be important for high-affinity binding. The C⑀3 domain also contains an attachment site (Asn-394) for N-linked carbohydrate that is conserved in other antibody classes (e.g. Asn-297 in the C␥2 domain of IgG Fc). Although the crystal structure did not reveal any contact with carbohydrate (6), an indirect effect of glycosylation upon receptor binding via stabilization of the polypeptide conformation cannot be ruled out. Indeed, an isolated C⑀3 domain expressed in Escherichia coli and lacking carbohydrate was found to be only partially folded (7) and NMR analysis of the same fragment indicates that it may have a molten-globule-like structure (8).The aim of this study was to determine the contributions of these two factors, dimerization and glycosylation of the C⑀3 domains, to the kinetics and affinity of Fc⑀RI binding. We have analyzed these effects using four variants of the Fc⑀3-4 fragment: (i) disulfide-linked and glycosylated (Fc⑀3-4); (ii) lacking only the disulfide link (redFc⑀3-4); (iii) lacking only glycosylation (deglyFc⑀3-4); and (iv) lacking both structural features (Fc⑀3-4⌬C). Fc⑀3-4 lacks the C⑀2 domains of the complete IgE Fc, but we have shown previously that not only does this fragment display a 1:1 stoichiom...

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Proregion ofBombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme

Yamamoto

Watabe

Kageyama

et al. 1999

Arch. Insect Biochem. Physiol.

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A cDNA encoding the proform of Bombyx cysteine proteinase (BCP) was expressed at a high level in Escherichia coli using the T7 polymerase expression system. The insoluble recombinant zymogen was solubilized and renatured by modifying a method applied to human pro‐cathepsin L. Like the natural BCP precursor, the recombinant proenzyme was spontaneously converted to an active proteinase at pH 3.75. A deletion in the central region of the propeptide resulted in much loss of the activity, suggesting that the propeptide is essential for proper folding during renaturation. In contrast, the renatured mature form of recombinant BCP was not active but regained activity by including the propeptide in the renaturing buffer, suggesting that the propeptide, acting as an intramolecular chaperone, promotes refolding of the associated proteinase domain into an active conformation. The mature form of natural BCP rapidly lost its activity at neutral pH, whereas its proform was stable. The mature enzyme retained some activity in the presence of the propeptide. Arch. Insect Biochem. Physiol. 42:167–178, 1999. © 1999 Wiley‐Liss, Inc.

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Active papain renatured and processed from insoluble recombinant propapain expressed in Escherichia coli

Cited by 51 publications

References 0 publications

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

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

Disulfide Linkage Controls the Affinity and Stoichiometry of IgE Fcϵ3–4 Binding to FcϵRI

Proregion ofBombyx mori cysteine proteinase functions as an intramolecular chaperone to promote proper folding of the mature enzyme

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