Purification of cathepsin B by a new form of affinity chromatography

Rich, D. H.; Brown, M A; Barrett, Alan J.

doi:10.1042/bj2350731

Cited by 81 publications

(46 citation statements)

References 20 publications

(17 reference statements)

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“…Preparation of Affinity Column-Glycyl-phenyalanyl-glycyl-semicarbazone (25 mg) was dissolved in 1 ml of methanol and combined with 5 ml of preswollen CNBr-activated aminohexyl-Sepharose 4B beads in a final volume of 50 ml of sodium carbonate buffer (100 mM, pH 8.0) as described previously (24). The mixture was rocked overnight at room temperature (RT), the beads were washed with 1 liter each of 50% methanol and deionized water, and unreactive sites were blocked with 6% ethanolamine for 4 h at RT.…”

Section: Methodsmentioning

confidence: 99%

“…Denatured globin was more rapidly hydrolyzed than hemoglobin in a reducing environment (not shown) and was slowly hydrolyzed under nonreducing conditions. DISCUSSION A principal cysteine protease of P. falciparum trophozoites was purified using an affinity chromatography protocol, which was adapted from a method for the purification of cathepsin B (24). Our purification provided, for the first time, a definitive amino-terminal sequence of this protease.…”

Section: Falcipain-2 a P Falciparum Trophozoite Hemoglobinasementioning

confidence: 99%

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Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

Shenai¹,

Sijwali²,

Singh

et al. 2000

Journal of Biological Chemistry

337

362

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Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mM glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.

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Section: Methodsmentioning

confidence: 99%

Section: Falcipain-2 a P Falciparum Trophozoite Hemoglobinasementioning

confidence: 99%

Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

Shenai¹,

Sijwali²,

Singh

et al. 2000

Journal of Biological Chemistry

337

362

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“…For purification of the protease to near homogeneity, we employed the affinity resin Sepharose-Phe-GlySc; however, we found that the addition of DTT to the enzyme prior to loading on the column as described (Rich et al, 1986) caused a loss in yield of up to 80%. To avoid this problem, we added enzyme and DTT to the resin simultaneously.…”

Section: High Yields Of Recombinant Rat Cathepsin B Have Been Reportementioning

confidence: 99%

“…avoid this problem, an affinity resin can be employed which, when the protein is eluted, allows for storage with the active site blocked (Rich et al, 1986).…”

Section: High Yields Of Recombinant Rat Cathepsin B Have Been Reportementioning

confidence: 99%

“…Sepharose-Phe-Gly-Sc resin was prepared as previously described (Rich et al, 1986) and packed into a 50 X 200 mm Waters AP-1 column. The column was equilibrated with Affinity A buffer (50 mM sodium phosphate pH 6.0, 1 .O mM EDTA) at a flow rate of 10 mL/min at 4 "C. The cathepsin B activity was loaded onto the column concomitantly with DTT to a final concentration of 2.0 mM.…”

Section: Expression Of Recombinant Human Cathepsin B In Yeastmentioning

confidence: 99%

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Characterization of recombinant human cathepsin B expressed at high levels in baculovirus

et al. 1998

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Abstract:The lysosomal cysteine protease cathepsin B has been studied intensely for many years because of its unique characteristics and its potential involvement in disease states. A reproducible, high yield expression system for active recombinant protein is key to biochemical and biophysical studies as well as rational drug design. Although several microbial and mammalian expression systems for recombinant human cathepsin B have been described, these have been limited by low or variable yields. Further, in some of these systems hyper-glycosylation of the enzyme near the active site affects its activity. We describe a baculovirus expression system and purification scheme that solve all of these problems. Yields of active, protected enzyme were reproducibly in excess of 25 mg/L. Since this protein was not hyper-glycosylated, it had greater activity than cathepsin B produced in yeast systems as indicated by a threefold increase in Kc,,. In addition, the biophysical properties of the baculovirus-expressed cathepsin B, as measured by dynamic light scattering, were more amenable to crystallographic study since the data indicated proteins of more uniform size. Therefore, this system for the production of recombinant human cathepsin B constitutes a major improvement in both quantity and quality over those previously reported. Further, we demonstrate that the manner of expression and purification of this enzyme has profound effects on its kinetic and physical parameters.

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Papain‐like Cysteine Proteases

Brὂmme

2000

CP Protein Science

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The name "cysteine protease" refers to the protease's nucleophilic cysteine residue that forms a covalent bond with the carbonyl group of the scissile peptide bond in substrates. The papain-like cysteine proteases, classified as the "C1 family" are the most predominant cysteine proteases. These proteases are found in viruses, plants, primitive parasites, invertebrates, and vertebrates alike. Mammalian papain-like cysteine proteases are also known as cathepsins. This unit discusses cathepsins, and their subcellular and tissue localization, catalytic mechanism, and substrate specificity. Several tables illustrate the properties of the various cathepsins.

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Purification of cathepsin B by a new form of affinity chromatography

Cited by 81 publications

References 20 publications

Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

Characterization of Native and Recombinant Falcipain-2, a Principal Trophozoite Cysteine Protease and Essential Hemoglobinase ofPlasmodium falciparum

Characterization of recombinant human cathepsin B expressed at high levels in baculovirus

Papain‐like Cysteine Proteases

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