6 research outputs found

    Molecular cloning, expression, and functional characterization of a cystatin from pineapple stem

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    A cDNA fragment encoding the cysteine protease inhibitor, cystatin, was cloned from pineapple (Ananas comosus) stem. This clone was constructed in a fusion vector and was easily over-expressed in Escherichia coli; satisfactory over-expression of non-fusion cystatin was achieved after an additional start codon was inserted prior to its coding sequence. Both recombinant cystatins were predominately found in the soluble fraction of the cell extract, and were demonstrated to be functionally active in a reverse zymographic assay. The fusion and non-fusion cystatins were separately purified to homogeneity via a His-tag or papain-coupling affinity column. Effective inhibitory activity against papain was detected with both the fusion and non-fusion cystatins with comparable K-i values of 1.18 x 10(-10) m and 9.53 x 10(-11) (M), respectively. The recombinant cystatins were found to be thermally stable up to 60 degreesC. Inhibition of the endogenous protease activity in minced fish muscle revealed that the recombinant pineapple cystatins might be an adequate stabilizer to prevent protein degradation during industrial food processing

    Cloning, functional expression and characterization of a phytocystatin gene from jelly fig (Ficus awkeotsang Makino) achenes

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    A cDNA clone encoding a phytocystatin was isolated and identified from about 300 expressed sequence tag (EST) clones in maturing jelly fig (Ficus awkeotsang Makino) achenes. This clone, named FaCYS, consists of 582 bp encoding 114 amino acids with a putative signal peptide. The predicted mature protein contains no cysteine and has a molecular mass of 10.8 kDa with an isoelectric point (pI) of 9.7. FaCYS constructed in nonfusion and fusion vectors were overexpressed in Escherichia coli as nonfusion and his-tagged recombinants, respectively. Both recombinants were found in the soluble fractions of the cell extracts. The purified nonfusion and his-tagged FaCYS exhibited papain inhibitory activity with similar Ki values of 2.7 x 10(-7) M and 2.4 x 10(-7) M, respectively. In addition, his-tagged recombinant proteins showed inhibitory activity toward human cathepsin B, cathepsin L and ficin with Ki values of 5.6 x 10(-7) M, 3.0 x 10(-8) M and 2.0 x 10(-7) M, but no inhibitory activity against stem bromelain. It was tolerant at a wide range of pH values and thermally stable up to 50 degrees C for 30 min. Furthermore, his-tagged FaCYS could arrest the fungal growth of Glomerella cingulata and Sclerotium rofsii

    A system for purification of recombinant proteins in Escherichia coli via artificial oil bodies constituted with their oleosin-fused polypeptides

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    An expression/purification system was developed using artificial oil bodies (AOB) as carriers for producing recombinant proteins. A target protein, green fluorescent protein (GFP), was firstly expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a linker sequence susceptible to factor Xa cleavage. Artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble recombinant protein, oleosin-Xa-GFP. After centrifugation, the oleosin-fused GFP was exclusively found on the surface of artificial oil bodies presumably with correct folding to emit fluorescence under excitation. Proteolytic cleavage with factor Xa separated soluble GFP from oleosin embedded in the artificial oil bodies; thus after re-centrifugation, GFP of high yield and purity was harvested simply by concentrating the ultimate supernatant. (C) 2004 Elsevier B.V. All rights reserved

    Cloning, functional expression, and characterization of cystatin in sesame seed

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    A cDNA fragment encoding cystatin, a cysteine protease inhibitor, was obtained from maturing sesame seeds. The clone was constructed in a nonfusion or fusion vector and then overexpressed in Escherichia coli. The recombinant cystatins were found in the soluble fraction of cell extract and were demonstrated to be functionally active in a reverse zymographic assay. The corresponding endogenous 22 kDa cystatin of low abundance in mature seeds was purified to homogeneity via a papain-coupling affinity column and confirmed by western blotting with antibodies against the recombinant cystatin. Both endogenous and recombinant cystatin proteins showed effective inhibitory activities against papain with K-i values of 7.89 x 10(-8) M and 2.77 x 10(-8) M, respectively. Immunodetection indicated that cystatin was specifically expressed in maturing seeds and rapidly degraded in germination. Accordingly, zymographic and inhibition analyses showed that sesame cystatin could not inhibit the de novo synthesized proteases in germinating seeds. It is suggested that sesame cystatin may play a role in the regulation of endogenous cysteine proteases during seed maturation and germination

    Method for bacterial expression and purification of sesame cystatin via artificial oil bodies

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    A method was developed for production of Sesame cystatin, a thermostable cysteine protease inhibitor. Sesame cystatin was first expressed in Escherichia coli as an insoluble recombinant protein fused to oleosin, a unique structural protein of seed oil bodies, by a short hydrophilic linker peptide. Stable artificial oil bodies were constituted with triacylglycerol, phospholipid, and the insoluble oleosin-cystatin fusion protein. After centrifugation, the oleosin-cystatin fusion protein was exclusively found in the artificial oil bodies. Proteolytic cleavage with papain, a cysteine protease effectively inhibited by cystatin, separated soluble cystatin from oleosin that was firmly embedded in the artificial oil bodies. After recentrifugation, papain that coexisted with cystatin in the collected supernatant was denatured by incubating at 55degreesC for 30 min. The insoluble denatured papain was removed by one more centrifugation, and the expressed cystatin of high yield and purity was harvested simply by concentrating the ultimate supernatant. Comparable inhibitory activity toward papain was observed between the expressed cystatin and the native one purified from sesame seeds. This method is presumably applicable to production of other protease inhibitors whose target proteases are economically available
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