Enhanced expression of chicken cystatin as a thioredoxin fusion form in Escherichia coli AD494(DE3)pLysS and its effect on the prevention of surimi gel softening |
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Authors: | Jiang Shann-Tzong Tzeng Shinn-Shuenn Wu Wun-Tsai Chen Gen-Hung |
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Institution: | Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China. sjiang@mail.ntou.edu.tw |
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Abstract: | The DNA encoding chicken lung cystatin was ligated into a thioredoxin-pET 23a+ expression vector and transformed into Escherichia coli AD494(DE3)pLysS. A high level of soluble recombinant thioredoxin-cystatin (trx-cystatin) was expressed in the cytoplasm of the E. coli transformant. As compared with recombinant cystatin (trx-free), a 38.7% increase of inhibitory activity in the soluble fraction was achieved by introducing the trx fusion protein. Trx-cystatin was purified to electrophoretical homogeneity by 3 min of heating at 90 degrees C and Sephacryl S-100 chromatography. The molecular mass of trx-cystatin was 29 kDa, which was the expected size based on its composition of recombinant trx (16 kDa) and chicken cystatin (13 kDa). The purified trx-cystatin behaved as a thermally stable and papain-like proteinase inhibitor comparable to either recombinant or natural chicken cystatins. The inhibitor could inhibit the gel softening of mackerel surimi. |
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