Chan M T, Chao Y C, Yu S M
Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China.
J Biol Chem. 1994 Jul 1;269(26):17635-41.
The 5' regulatory region and putative signal sequence of a rice alpha-amylase gene, alpha Amy8, was fused to a bacterial gene encoding beta-glucuronidase (GUS) and introduced into rice, tobacco, and potato via Agrobacterium-mediated transformation systems. Expression of this chimeric gene in suspension cells of transgenic plants was suppressed by the presence of sucrose in the medium and induced by its absence. Induction or suppression of GUS expression in transgenic rice could be reversed by the deprivation or replenishment, respectively, of sucrose in the medium. The expressed GUS fusion protein was translocated to the endoplasmic reticulum, modified by glycosylation, and secreted into the culture medium of transgenic cells. In the presence of a glycosylation inhibitor, tunicamycin, the enzymatically active form of GUS was assembled in the endoplasmic reticulum. The yield of GUS secreted by transgenic cells was estimated to be as high as 40% of total secreted proteins. The reversible induction of the alpha-amylase promoter in culture cells by sugar level in the medium provides an excellent inducible expression system for basic research in plant science. Combination of the alpha-amylase promoter and signal sequence also offers a novel approach for large scale production of low cost, easily purified, secreted recombinant proteins.
将水稻α-淀粉酶基因α Amy8的5'调控区和假定信号序列与编码β-葡萄糖醛酸酶（GUS）的细菌基因融合，并通过农杆菌介导的转化系统导入水稻、烟草和马铃薯。该嵌合基因在转基因植物悬浮细胞中的表达在培养基中存在蔗糖时受到抑制，在无蔗糖时被诱导。通过分别剥夺或补充培养基中的蔗糖，转基因水稻中GUS表达的诱导或抑制可以被逆转。表达的GUS融合蛋白被转运到内质网，经糖基化修饰后分泌到转基因细胞的培养基中。在糖基化抑制剂衣霉素存在的情况下，GUS的酶活性形式在内质网中组装。转基因细胞分泌的GUS产量估计高达总分泌蛋白的40%。培养基中糖水平对培养细胞中α-淀粉酶启动子的可逆诱导为植物科学基础研究提供了一个优良的诱导表达系统。α-淀粉酶启动子和信号序列的组合也为大规模生产低成本、易于纯化的分泌型重组蛋白提供了一种新方法。
