Jiang Shann-Tzong, Tzeng Shinn-Shuenn, Wu Wun-Tsai, Chen Gen-Hung
Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan 202, Republic of China.
J Agric Food Chem. 2002 Jun 19;50(13):3731-7. doi: 10.1021/jf020053v.
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.
编码鸡肺半胱氨酸蛋白酶抑制剂的DNA被连接到硫氧还蛋白-pET 23a+表达载体中，并转化到大肠杆菌AD494(DE3)pLysS中。在大肠杆菌转化体的细胞质中高水平表达了可溶性重组硫氧还蛋白-半胱氨酸蛋白酶抑制剂（trx-半胱氨酸蛋白酶抑制剂）。与重组半胱氨酸蛋白酶抑制剂（无trx）相比，通过引入trx融合蛋白，可溶性部分的抑制活性提高了38.7%。通过在90℃加热3分钟和Sephacryl S-100层析，将trx-半胱氨酸蛋白酶抑制剂纯化至电泳纯。trx-半胱氨酸蛋白酶抑制剂的分子量为29 kDa，根据其重组trx（16 kDa）和鸡半胱氨酸蛋白酶抑制剂（13 kDa）的组成，这是预期的大小。纯化的trx-半胱氨酸蛋白酶抑制剂表现为一种热稳定的、类似于木瓜蛋白酶的蛋白酶抑制剂，与重组或天然鸡半胱氨酸蛋白酶抑制剂相当。该抑制剂可以抑制鲭鱼鱼糜的凝胶软化。
