Expression of Soluble Thioredoxin Fused-Carp (Cyprinus carpio) Ovarian Cystatin in Escherichia coli |
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| Authors: | S.-S. Tzeng G.-H. Chen S.-T. Jiang |
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| Affiliation: | Authors Tzeng, Chen, and Jiang are with the Dept. of Food Science, National Taiwan Ocean Univ., Keelung, Taiwan 202, ROC. Direct inquiries to author Jiang (E-mail: ). |
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| Abstract: | ABSTRACT: A DNA-encoding thioredoxin-carp ovarian cystatin (trx-cystatin) was ligated into pET-23a(+) and transformed into Escherichia coli AD494(DE3). High level of soluble recombinant trx-cystatin, expressed in E. coli was purified by 5 min of heating at 70 °C, Q-Sepahrose HP, and Sephacryl S-100 HR chromatographs. Its molecular mass was 28 kDa. It could be cleaved into a recombinant thioredoxin (16 kDa) and a mature carp ovarian cystatin (12 kDa) by enterokinase. The 12-kDa mature carp ovarian cystatin was further purified by FPLC Superdex 75 chromatography. Both recombinant trx-fused and carp ovarian cystatins were thermostable proteins and exhibited papain-like protease inhibition activity comparable to the wild-type cystatin. |
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| Keywords: | carp ovary trx-cystatin E. coli AD494(DE3) cysteine proteinase inhibitor purification |
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