Shyu Douglas J H, Chou Wing-Ming, Yiu Tien-Joung, Lin Coney P C, Tzen Jason T C
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan 40227, Republic of China.
J Agric Food Chem. 2004 Mar 10;52(5):1350-6. doi: 10.1021/jf034989v.
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.
从成熟芝麻种子中获得了一个编码半胱氨酸蛋白酶抑制剂胱抑素的cDNA片段。该克隆构建于非融合或融合载体中，然后在大肠杆菌中过表达。重组胱抑素存在于细胞提取物的可溶部分，并在反向酶谱分析中被证明具有功能活性。成熟种子中低丰度的相应内源性22 kDa胱抑素通过木瓜蛋白酶偶联亲和柱纯化至同质，并通过用抗重组胱抑素的抗体进行蛋白质印迹法确认。内源性和重组胱抑素蛋白对木瓜蛋白酶均表现出有效的抑制活性，其抑制常数（K(i)）值分别为7.89×10⁻⁸ M和2.77×10⁻⁸ M。免疫检测表明，胱抑素在成熟种子中特异性表达，并在萌发过程中迅速降解。因此，酶谱分析和抑制分析表明，芝麻胱抑素不能抑制萌发种子中新合成的蛋白酶。提示芝麻胱抑素可能在种子成熟和萌发过程中对内源性半胱氨酸蛋白酶的调节中发挥作用。
