Expression of human cathepsin B protein in <i>Escherichia coli</i>

Chan, Marion Man Ying; Fong, Dunne

doi:10.1016/0014-5793(88)80920-7

Cited by 15 publications

(15 citation statements)

References 17 publications

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“…Northern blot analysis of the isolated cathepsin B mRNA revealed two distinct cathepsin B transcripts (4.1 and 2.2kb) in all specimens, including normal brain tissue (Figure 4). The sizes of these transcripts are similar to those published [17,18]. The amounts ofcathepsin B m RNA were markedly higher in the glioblastoma samples and moderately higher in the anaplastic astrocytomas than in normal brain tissues and low-grade glioma tissue samples.…”

Section: Northern Blottingsupporting

confidence: 71%

“…Total tissue RNA (20/~g) from each sample was electrophoreses in formaldehyde containing 1% agarose gels and transferred to Hybond membranes (Amersham Corp., Arlington Heights, IL) by capillary action using 10 x SSC buffer. The membranes were fixed by baking at 80°C for 2 h, and the blots were probed at 42°C with random-primed 32p-labeled cathepsin B cDNA probes [17,18]. The probes were labeled with g-[32p]-dCTP (6000Ci/mmol) using a randomprimed labeling kit (Boehringer Mannheim Corp., Indianapolis, IN).…”

Section: Rna Extraction and Northern Blottingmentioning

confidence: 99%

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Overexpression and localization of cathepsin B during the progression of human gliomas

et al. 1995

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Degradation of the extracellular matrix is a prerequisite for acquisition of the invasive phenotype. Several proteinases released by invading tumor cells appear to participate in the focal degradation of extracellular matrix proteins. Using an enzyme-linked immunosorbent assay, enzymatic assays, Western and Northern blotting techniques, we determined whether increased levels of the cysteine protease cathepsin B correlated with the progression and invasion of human gliomas. The amount of cathepsin B activity and protein content were highest in glioblastomas, lower in anaplastic astrocytomas and lowest in normal brain tissue and low-grade gliomas. There were significantly higher amounts of M(r) 25,000 and 26,000 bands in glioblastoma and anaplastic astrocytoma than in normal brain and low-grade glioma tissue extracts as determined by Western blotting with anti-cathepsin antibodies. In addition, cathepsin B transcripts were overexpressed in anaplastic astrocytoma (about two- to three-fold), in glioblastoma (about eight- to 10-fold), compared with normal brain tissue and low-grade glioma. Immunohistochemical staining for cathepsin B showed intense immunoreactivity in tumor and endothelial cells of glioblastomas and anaplastic astrocytomas but only weak immunoreactivity in low-grade glioma and normal brain tissues. Therefore, we conclude that cathepsin B expression is greatest in highly malignant astrocytomas, especially in glioblastomas, and is correlated with the malignant progression of astrocytomas.

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Section: Northern Blottingsupporting

confidence: 71%

Section: Rna Extraction and Northern Blottingmentioning

confidence: 99%

Overexpression and localization of cathepsin B during the progression of human gliomas

et al. 1995

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“…It was reported previously (Chan and Fong, 1988) that the expression of cathepsin B in E. coli BL21(DE3) strain failed, probably because the basal level of T7 RNA polymerase activity had promoted some transcription of the target gene in the uninduced cell. To avoid the problem of cytotoxicity of procathepsin B we used the E. coli BL21(DE3)-pLysS strain, which contains the pLysS plasmid coding for T7 lysozyme, a natural inhibitor of T7 RNA polymerase (Studier et al, 1990).…”

Section: Discussionmentioning

confidence: 97%

“…Expression of rat and mouse cathepsin B (Mort et al, 1988) and of human cathepsin B (Chan and Fong, 1988) in Escherichia coli did not result in an active enzyme although cathepsin B could be detected immunologically in both cases. Rat procathepsin B expressed in Saccharomyces cerevisiae was heterogeneously glycosylated (Hasnain et al, 1992) ; the problem was circumvented by changing the serine at position 115 to alanine to eliminate the consensus sequence for N-linked oligosaccharide substitution, -Asn-Gly-Ser-.…”

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confidence: 99%

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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“…For the four groups of proteinases -the serine, cysteine, aspartic, and metallo-typesmany proteinase inhibitors are known (Barrett and Salvesen 1986). We are interested in the lysosomal cysteine proteinase cathepsin B because it has been implicated in cancer metastasis (Fong et al 1986;Chan and Fong 1988;Moin et al 1989). Our study of cathepsin B has guided us to the cystatins.…”

Section: Introductionmentioning

confidence: 99%

The human kininogen gene (KNG) mapped to chromosome 3q26-qter by analysis of somatic cell hybrids using the polymerase chain reaction

1991

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Kinins, peptide products of kininogens, may be involved in hypertensive and diabetic diseases, and inflammatory disorders. The human kininogen gene (KNG) has been mapped to chromosome 3, using a panel of human-hamster somatic cell hybrids by polymerase chain reaction of hybrid DNA with gene-specific primers. KNG was further assigned to 3q26-3qter, using DNA from a second panel of chromosome 3 deletion mapping cell hybrids.

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Expression of human cathepsin B protein in Escherichia coli

Cited by 15 publications

References 17 publications

Overexpression and localization of cathepsin B during the progression of human gliomas

Overexpression and localization of cathepsin B during the progression of human gliomas

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

The human kininogen gene (KNG) mapped to chromosome 3q26-qter by analysis of somatic cell hybrids using the polymerase chain reaction

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