Jheng Yi-Jyun, Tsai Wei-Yi, Chen Kuo-Hsuan, Lin Kuo-Wei, Chyan Chia-Lin, Yang Ching-Chi, Lin Kuo-Chih
Department of Life Science, National Dong Hwa University, Hualien County 974, Taiwan, ROC.
Protein Expr Purif. 2012 Sep;85(1):77-85. doi: 10.1016/j.pep.2012.07.001. Epub 2012 Jul 14.
Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins.
薯蓣球蛋白是山药块茎中的主要贮藏蛋白，具有生化和免疫调节活性。为了研究薯蓣球蛋白在生物医学研究中的潜在应用，我们分别在大肠杆菌中表达了来自日本薯蓣和伪日本薯蓣的薯蓣球蛋白基因Dj-dioA3和Dp-dioA2，常规情况下每升大肠杆菌培养物可获得约15毫克蛋白质（毫克/升），即30毫克/升的重组日本薯蓣球蛋白A3和4至8毫克/升的重组伪日本薯蓣球蛋白A2。蛋白质免疫印迹分析表明，两种重组薯蓣球蛋白都含有与天然薯蓣球蛋白抗原性相似的表位。二硫苏糖醇（DTT）处理后再用单溴代双马来酰亚胺（mBBr）染色的结果表明，两种重组薯蓣球蛋白与天然薯蓣球蛋白一样，在半胱氨酸（28）和半胱氨酸（187）残基之间含有分子内二硫键。圆二色光谱分析结果表明，重组薯蓣球蛋白的二级结构含量与其相应的天然薯蓣球蛋白高度相似。两种重组薯蓣球蛋白与天然薯蓣球蛋白一样，都表现出1,1-二苯基-2-苦基肼（DPPH）自由基清除活性和Toll样受体4信号传导活性，并刺激巨噬细胞对大肠杆菌的吞噬作用。总体而言，我们的结果表明，可以很容易地从大肠杆菌中纯化出大量的重组薯蓣球蛋白，并且这些重组薯蓣球蛋白适用于未来对薯蓣球蛋白生物医学功能的研究。
